USE STABLE ISOTOPES TO INVESTIGATE MICROBIAL H
2
 
AND N
2
O PRODUCTION
 
By
 
Hui Yang
 
 
 
 
 
A DISSERTATION
 
Submitted to
 
Michigan State University
 
in partial fulfillment of the requirements
 
for the degree of 
 
Biochemistry and Molecular Biology 
-
 
Doctor of Philosop
hy
 
2015
 
 
 
ABSTRACT
 
USE STABLE ISOTOPES TO INVESTIGATE MICROBIAL H
2
 
AND N
2
O PRODUCTION
 
By
 
Hui Yang
 
Stable isotopes can be a useful tool in studying the basic processes involved in enzymatic 
catalysis.  Isotope effects are quantifiable values related to the s
ubstitution of isotopes.  It derives 
from the difference in zero
-
point energies.  In contrast to non
-
catalyzed reactions, enzyme
-
catalyzed reactions involve multiple steps, the overall isotope effects are the sum of the isotope 
effects in each step.  There
 
are two major kinds of isotope effects, equilibrium isotope effects 
(EIEs) and kinetic isotope effects (KIEs).   Each of them can provide us insights into different 
states in the reaction.  This thesis describes several researches related to using the sta
ble isotopes 
to study microbial metabolism.  
 
It is demonstrated in Chapter 2 that a method is developed for the measurement of H isotope 
fractionation patterns in hydrogenases.  After the development of the method, a detailed study of 
the H
2
 
metabolism ca
talyzed by different hydrogenases is presented in Chapter 3.  The methods 
developed in hydrogenase studies were deployed in nitric oxide reductase
-
catalyzed N
2
O 
production studies, which is described in Chapter 4.  
iii
 
TABLE OF CONTENTS
 
 
 
LIST OF TABLES



.
.v
i
 
LIST OF FIGURES


...
v
i
i
 

1
 

1
 
Nomenclature

.2
 
T
he basics of isotope effect


3
 
E
nzyme
-
catalyzed versus uncatalyzed kinetic isotope effects

.10
 
M
agnitude of the observed isotope effect in enzyme
-
catalyzed reactions and the commitment 
to catalysis

....10
 
A
pplications of stable isot
opes in chemistry and biology

...13
 
A
pplications of stable isotopes in biogeochemistry

.15
 
C
onclusion

...18
 
BIBLIOGRAPHY

2
0
 
CHAPTER 2

..
2
3
 
U
SING GAS CHROMATOGRAPHY
-
ISOTOPE RATIO MASS SPECTROMETRY TO 
DETERMINE THE FRACTIONATOIN FACTOR FOR H
2
 
PRODUCTION BY 
HYDROGENASES
 

2
4
 

2
5
 


2
7
 
H
2
 
Evolution Assay

....27
 
IRMS Measurements

.29
 
Measurement of 

2
H
2
O

.

.36
 
D
ata Analysis

.36
 

3
7
 
Calculations for determining the fractionation factor for H
2
 
evolution

.40
 
P
otential Applications

42
 

44
 
BIBLIOGRAPHY


4
6
 


51
 
MEASURING H ISOTOPES TO DETERMINE THE FRACTIONATION FACTORS FOR 
[NiFe]
-
 
AND [FeFe]
-
HYDROGENASES
 

5
2
 

5
3
 

5
6
 
Enzyme 
pr

...56
 
H
2
 
evolution assay

..57
 
H
2
 
uptake assay

..64
 
iv
 
H
2
 
exchange assay

..65
 
Standardization and notation

..65
 


66
 
Isotopic fractionation associated with H
2
 
evolution

..66
 
I
sotopic fractionation associated with H
2
 
consumption

.

72
 
I
sotopic fractionation associated with H
2
-
H
2
O exchange

..

74
 


75
 
APPENDIX

..

8
1
 
BIBLIOGRAPHY

8
4
 

89
 
ISOTOPIC FRACTIONATION BY A FUNGAL P450 NITRIC OXIDE REDUCTASE 
DURING THE PRODUCTION OF N
2
O
 

90
 

91
 

95
 
E
xpression and purification of P450nor

....95
 
P450nor activity assay

...95
 
IR
MS measurements

..96
 
I
sotope value notations

..97
 
C
alculations for determining enrichment factor (

) and kinetic isotope effects (KIE)

..98
 


10
3
 

15
N and 

18
O analyses 
for N
2
O produced by 
H. capsulatum
 
P450nor

...103
 
Site preference values for N
2
O produced by 
H. capsulatum
 
P450nor

.....110
 


1
12
 
F
ractionation during reaction of NO by P450nor

112
 
I
mplications
 
for the reaction pathways of NO reaction by P450nor

...114
 
I
mplication for isotope source tracing of N
2
O production

..

.117
 
BIBLIOGRAPHY


1
21
 

12
9
 
CONCLUSIONS


9
 
BIBLIOGRAPHY

3
4
 
 
 
v
 
LIST OF TABLES
 
 
 
Table 1
.
  


...3
 
Table 
2:
 
 

2
H values of H
2
 
obtained for the Oztech H
2
 
reference gas



...3
2
 
Table 
3:
 
 

2
H values of MSU internal laboratory standards measured over three days as a 
function of the amount of H
2
 
injected


.3
3
 
Table 
4:
 
 
Isotope ratio of H
2
 
produced by 
Desulfovibrio fructosovorans 
hydrogenase


.3
8
 
Table 
5
. 
 

2
H data
 
for production of H
2
 
by different hydrogenases
.


..5
8
 
Table 
6:
  

2
 
production catalyzed by different [NiFe]
-
 
and [FeFe]
-
hydrogenases


.6
8
 
Table 
7: 
 
Fractionatio

2
 
consumption (i.e. 
oxidation) catalyzed by different [NiFe]
-
 
and [FeFe]
-
hydrogenases.

..

...7
4
 
Table 
8:
  

replica
tes of 
H. capsulatum
 
P450nor


10
6
 
 
 
 
vi
 
LIST OF FIGURES
 
 
 
Figure 1
:
  
A simplified characterization of a hypothetical reaction coordinate representing 
progress along a reaction pathway
....

...
...
4
 
Figure 2
:
  
A picture depictin
g the zero
-
point energy and its causal relationship with the kinetic 
isotope effects


..5
 
Figure 3
:
  
A picture depicting the potential energy curves for the normal equilibrium isotope 
effect (3A), the normal kinetic isotope effect
s (3B), and the inverse kinetic isotope 
effect (3C)

...


7
 
Figure 4
:
  
A simple reaction illustrating a hypothetical progression from the substrate to the 
product


...12
 
Figure 5
:
 
 
Two proposed reaction 
mechanisms for the aspartate transcarbamoylase catalyzed 
reaction



..14
 
Figure 6
:
  
Changes of isotope ratios of the substrate, the instantaneous product, and the 
accumulated product in both open and closed systems

.1
8
 
Figure 
7: 
 
Schematic representation of the GC
-
IRMS system designed for the stable isotopic 
analysis of gaseous H
2


...3
1
 
Figure 
8:
 
 

2
H by the GC
-
IRMS system

.......

.3
6
 
Figure 
9
:
  
Structures of the active sites in [NiFe]
-
 
and [FeFe]
-
hydrogenases

...5
4
 
Figure 
10:
  

2
H measurements of H
2
-
H
2
O exchange reactions catalyzed by different 
hydro
genases over the course of 180 min

..6
4
 
Figure 
11:
  
Isotope ratio of H
2
 
produced by different hydrogenases


.6
7
 
Figure 
12:
 
 
Measured H
2
 
production rate over the course of the analyses


...

.6
9
 
Figure 
13:
  
Relationship between r

2
H of the H
2
 
produced in H
2
 
evolution 
reaction tested


.
71
 
Figure 
14:
  
Relationships between isotope ratio of headspace H
2
 

2
) and the fraction of H
2
 
consumed (
f
) for 4 different hydrogenases tested


...7
3
 
Figure 
15:
  

2
H of the H
2
 
produced by [FeFe]
-
hydrogenases versus 
[NiFe]
-
hydrogenases


..7
6
 
Figure 
16:
  
Multiple sequence alignment of P450nor(s)




...9
3
 
vii
 
Figure 
17:
  
Best fit line of the Ra

15
N in N
2
O produced by P450nor

..
9
9
 
Figure 
18:
  

18
O in N
2
O produced by P450nor


100
 
Figure 
19:
  

the th
ree biological replicates


...101
 
Figure 
20:
  
Best fit line of the Rayleigh equation to site preference (SP) values


102
 
Figure 
2
1
:
  
N
2
O production by 
H. capsulatum
 


103
 
Figure
 
22
:
 
 

15

18
O o
f N
2
O produced by P450nor as a function of the fraction of NO 
reduced (1
-
f


.10
5
 
Figure 
2
3:
 
 
Quantification of N
2
O in the headspace in both the presence and absence of 


.10
7
 
Figure 
2
4:
  
Character
ization of the N
2
O isotope values to test for the presence of NO
-
N
2
O 


...10
8
 
Figure 
2
5
:
  

15
N

 

15
N

 
of N
2
O produced by as a function of the fraction of NO reduced (1
-
f


1
10
 
Figure 
2
6
:
  
Site preference (SP) values of N
2
O versus the fraction of NO reduced (1
-
f


1
11
 
Figure 
2
7
:
  
An energy diagram depicting t
he proposed binding of the first NO to the P450nor 


1
15
 
 
 
1
 
 
 
 
 
 
 
 
 
CHAPTER 1
 
INTRODUCTION
 
 
2
 
In 1929 Giauque et al. discovered that the oxygen atom has two heavy isotopes, 
17
O and 
18
O.
1
  
This finding led to speculation that hydrogen m
ust also have a heavy isotope based on the 
accurate determination of the molecular weight of H
2
O.
2
  
Indeed, in 1932 Urey et al. reported 
spectroscopic evidence of deuterium for the first time.
3
  
These experiments served as the 
founda
tion for the discovery of pure heavy water
4
 
and the formation of the theory of zero
-
point 
energy and isotope effects,
5
 
which set the cornerstone for an explosion of research into the usage 
of isotopes and ultimately a Nobel Prize for Urey.  
 
Isotopes are atoms of the same element that behave similarly in chemical reactions but ha
ve a 
different mass due to the different numbers of neutrons they are carrying.  Stable isotopes are 
valuable tools in chemistry, biology, and biogeochemistry as tracers, internal standards, and 
mechanistic probes.
6
  
Many of the studies involve using different isotopes a
t enriched levels or 
radioactive isotopes, which are easy to detect.  However, as the sensitivity of mass spectrometers 
increases, one can perform experiments using stable isotopes at natural abundance levels to study 
chemical and biochemical problems.  In
 
this chapter, the theory of isotope effects is briefly 
introduced, followed by a summary of key applications of stable isotopes in various research 
applications.
 
Nomenclature
 
The abundance of low
-
mass isotopes (H, O, N and S) in a compound is normally rep
orted as the 

equation (Equation 1):
7
 

x
/R
s
 
-
 
1)*1000                                                                                                                      
 
(1)
 
3
 
where R
x
 
and R
s
 
are the ratios of the heavy to light isotope in the sample and standa
rd, 
respectively.  Table 1 lists the isotopes used in our research and their corresponding standards. 
 
 
Table 1
:
  
The isotopes and their respective standards used in our studies.  
 
Isotope
 
Ratios measured
 
Standard
 
Absolute abundance 
ratio (R) of standard
 
H
 
2
H/
1
H
 
VSMOW
a
 
1.5575*10
-
4
 
N
 
15
N/
14
N
 
Atmospheric N
2
 
3.677*10
-
3
 
O
 
18
O/
16
O
 
VSMOW
a
 
2.0052*10
-
3
 
a
VSMOW (Vienna Standard Mean Ocean Water) is used as a standard for both H and O 
isotopes.
8
  
 
 
The basics of isotope effects
 
In a chemical reaction, the phenomenon of isotopically
-
substituted molecules reacting at 
different rates and therefore fractionating
1
 
is known as the isotope effect.  Although substitution 
of one isotope with
 
the other changes the reaction rate, it will not alter the reaction coordinate 
(Figure 1), whose curvature depicts the potential energies of the substrate, transition state(s), and 
product.  Isotope effects arise because of the nature of all chemical bond
s, which can be 
described as harmonic oscillators.  The energy of a chemical bond is equal to:
 
                                        
                  
 
1
 
F
ractionat
ion is the 
change 
in the 
relative proportions of various isotopes in molecules 
during
 
a reaction.  
 
4
 
E = ½(


 
 
 
 
 
 
 
 
 
 
 
(2)
 
where 

 

h

vibrational frequency of a bond is inve
rsely proportional to the square root of the reduced mass 


 



) where
 

1
m
2
/(m
1
+m
2
)
 
 
 
 
 
 
 
 
 
 
(3)
 
 
 
Figure 1
:
  
A simplified characterization of a hypothetical reaction coordinate representing 
progress along a reaction pathway. 
 
Isotopic s
ubstitution does not change the shape of the 
reaction coordinate.  
 
 
Because heavy isotopes give rise to larger reduced masses, which in turn lead to lower 
vibrational frequencies, chemical bonds with heavy isotopes will have lower zero
-
point energies 
(ZPE
, Figure 2).
9
  
It is this difference in zero
-
point energy that is the basis for
 
isotope effects.
 
5
 
 
Figure 2
:
  
A picture depicting the zero
-
point energy and its causal relationship with the 
kinetic isotope effects.
  
2A shows the different quantum energy states of a typical C
-
H bond; the 
lowest possible energy level is called zero
-
poin
t energy.  In 2B, the hypothetical cleavage of a C
-
H and C
-
D bond is compared to highlight that the difference in activation energies between the 
two events is a result of the difference in zero
-
point energies of the two bonds.  
 
6
 
There are two fundamentall
y different types of isotope effects 

 
equilibrium isotope effects and 
kinetic isotope effects.  Equilibrium isotope effects (EIEs) are the differences between R
substrate
 
and R
product
 
and arise from the differences in zero
-
point energies between the substr
ate and the 
product (Figure 3A), which, in turn, result in a change in the ratio of the equilibrium constants 
for the heavy and light isotopes (Equation 4).
10
  
Conversely, kinetic isotope effects (KIEs) are 
caused 
by the differences of zero
-
point energies between the ground state and the transition state, 
which give rise to the change in rate constants of the heavy and light isotopes (Equation 5).
7
 
EIE = 
K
light
/
K
heavy
 
 
 
 
 
 
 
 
 
 
(4)
 
KIE = 
k
light
/
k
heavy
 
 
 
 
 
 
 
 
 
 
(5)
 
where 
K
light
 
and 
K
heavy
 
are the equilibrium constants of the light and heavy isotopes, respectively, 
and 
k
li
ght
 
and 
k
heavy
 
are the rate constants for the light and heavy isotopes, respectively.  Because 
the KIE and EIE are closely associated with the transition states and intermediates, isotope 
effects can be a valuable tool in elucidating the reaction mechanism
.  It should be noted, however, 
that due to the possibility of atom tunneling (especially for hydrogen), some isotope effects 
cannot be explained simply by classical transition state theory.  For additional information on 
hydrogen atom tunneling, please re
fer to a review published by Kohen et al.
11
  
 
7
 
 
Figure 3
:
  
A picture depicting the potential energy curves for the normal equilibrium 
isotope effect (3A), the normal kinetic isotope effects (3B), and the inverse kinetic isotope 
effect (3C). 
 
Zero
-
point energies are represented on the graph of hypothetical reaction 
8
 
coordinates.  When the difference of zero
-
point energies in the ground state is larger than those 
in the transition state, there is a normal kinetic isotope effect.  Conversely, an i
nverse kinetic 
isotope effect is observed when the difference of zero
-
point energies in the transition state is 
larger than those in the ground state.  An equilibrium isotope effect is generated due to a 
difference in the zero
-
point energies between the su
bstrates and the products. 
 
 
Because isotope effects are calculated as a ratio, they are often compared to 1.  If a kinetic 
isotope effect value is larger than 1 (i.e. the light isotope reacts more quickly than the heavy 
isotope, and the substrate therefor
e becomes enriched in heavy isotopes over time), it is 

observed in both biochemical and chemical reactions.  In some instances, however, the isotope 
effect value can
 
be smaller than 1 (i.e. the heavy isotope reacts more quickly than the light 
isotope, and the substrate therefore becomes depleted in heavy isotopes over time), in which case 

 
The presence of in
verse kinetic isotope effects may seem counterintuitive since it implies that a 

have the lower zero
-
point energy.  This apparent conundrum can be easily explaine
d, however, 
by recognizing that the magnitude and type (i.e. normal or inverse) of the isotope effect is 

state.  In other words, any factors that contribute 


(for EIEs), will affect the magnitude (and potentially the type) of isotope effect.  For example, 
9
 
the steepness of
 
an energy potential well in a reaction coordinate is determined by the bond 
strength between the two connected atoms, with stronger bonds leading to steeper wells.  

nd order, 
and therefore the bond strength, will have a significant impact on the value of the isotope effect.
 
In the field of biogeochemistry, people often use different terminology to describe the isotopic 

 

widely used terms.  The fractionation factor is calculated as the ratio of the isotope ratio of the 
product versus the substrate (Equation 6).  
 

product
/R
substrate
                                        
                                                                                  
(6)
 
The enrichment factor is a highly related expression and is simply the difference of the isotope 
ratios between the product and substrate (Equation 7).  
 

product
 
-
 

substrate
                                                                                                                          
(7)
 
The fractionation factor and enrichment factor are mathematically related (Equation 8), 
 

                                                                                                               
(8)
 
and both terms are quantitatively related to kinetic isotope effects, as shown in Equation 9, 
 

-
1
                                   
                                                                                                   
(9)
 
where KIE is defined as the ratio of the rate constants of the light and heavy isotopes, i.e. 
k
L
/
k
H
.  
 
 
 
10
 
Enzyme
-
catalyzed versus uncatalyzed kinetic isot
ope effects
 
Uncatalyzed reactions often occur in a single step, while enzyme
-
catalyzed reactions are always 
composed of multiple steps.  The steps involved in enzyme catalysis include: (1) the binding of 
substrate to the enzyme, (2) conversion of substrate
 
into product, and (3) release of the product 
from the enzyme active site.  In addition, the conversion of substrate into product can itself be a 
multistep process involving multiple transition states and intermediates.
12
  
In this process, a 
complex enzymati
c reaction can be thought of as a series of individual steps, with each step 
having its own reactants, products, and activation energies.  In theory, this can lead to KIEs and 
EIEs for each individual step.  The observed KIE and EIE for a complex reaction 
is a 
combination of all of the individual steps.  For uncatalyzed reactions, the rate determining step, 
which dictates the magnitude of the KIE, is determined by the nature of the bond
-
breaking and/or 
bond
-
forming steps.  For enzyme
-
catalyzed reactions, ho
wever, processes other than the actual 
substrate bond
-
breaking or bond
-
forming steps (such as substrate binding or product release) can 
be rate
-
determining.  In addition, by definition catalyzed reactions have lower energy transition 
states than uncatalyze
d reactions.  For this reason, enzyme
-
catalyzed reactions typically have a 
different kinetic isotope effect compared to their corresponding uncatalyzed reactions.
 
Magnitude of the observed isotope effect in enzyme
-
catalyzed reactions and 
the commitment to 
catalysis
 
Because enzyme
-
catalyzed reactions are complex, the isotope effects measured are often not 
controlled by one single rate
-
determining step, but rather by several partially rate
-
determining 
steps.  In other words, there is a difference between the 


11
 
effects caused only by the actual enzyme
-
catalyzed step).  While the observed fractionation 
factor can be affected by pro
cesses other than the isotopically sensitive step and display a broad 
range of values depending on different experimental conditions (e.g. substrate concentrations, 
experimental designs, etc.), the intrinsic fractionation factor represents the full isotope
 
effect 
imposed on the isotopically sensitive step and is a single value.  Therefore, to interpret an 
enzyme mechanism in terms of transition
-
state theory, it is important to separate the intrinsic 
isotope effect from the observed isotope effect.
 
According
 
to Northrop,
13
 
the observed isotope effect and intrinsic isotope effect are 
mathematically related.  Two of the important parameters that can affect the mathematical 
relationship between observed and intrin
sic isotope effects are the forward commitment to 
catalysis (
C
f
) and the reverse commitment to catalysis (
C
r
).  In this representation, 
C
f
 
represents 
the tendency of the enzyme complex to continue forward to product and 
C
r
 
represents the 
tendency of the en
zyme complex following the isotopically sensitive step to follow reverse 
catalysis toward reactants.
14
 
There are two general methods to measure isotope effects.  In the c
lassical method, substrates are 
largely isotopically substituted and 
V
max
/
K
m
 
(maximum reaction rate versus Michaelis constant) 
values for different isotopic species are compared.  In this thesis, I employ a different method in 
which isotope fractionation/d
istribution in the products and reactants are measured in systems 
containing near natural abundance isotope ratios.  This is a competitive method in which the 
substrate is trace
-
labeled so there is limited isotope effect on reaction rate if observed direct
ly, 
and the isotope effects are obtained by monitoring the rate of isotopic enrichment (or depletion) 
in the substrate or product during the initial phase of the reaction.  For the traditional method, the 
isotope effect observed is proportional to the exte
nt of how rate
-
limiting the isotopically 
12
 
sensitive step is, as the commitment of catalysis will not alter the overall rate of the reaction.  
However, for the competitive method presented in this thesis, the commitment to catalysis factor 
might affect the o
bserved isotope effect (Figure 4), because the unpreferred isotopic species can 
be accumulated at the isotopically sensitive step and go through the reverse catalysis, therefore 
changing the observed results.  In essence, depending on the value of commitme
nt to catalysis, 
the observed results in the latter case could be a combination of the forward kinetic isotope effect, 
the reverse kinetic isotope effect, and the equilibrium isotope effect.  If the commitment to 
catalysis of the reaction is 1 (i.e. the re
action is irreversible), there is no isotope effect observed 
in the competitive method.  If, however, the commitment to catalysis is 0 (i.e. the reaction is 
fully reversible), the observed isotope effect is equivalent to the intrinsic isotope effect in the
 
competitive method utilized in this thesis.  
 
 
 
Figure 4
:
  
A simple reaction illustrating a hypothetical progression from the substrate to 
the product.
  
In this figure, the commitment to catalysis step is before the isotopically sensitive 
step, which wil
l lead to different observed isotope effects depending on the methods used.  
 
13
 
Applications of stable isotopes in chemistry and biology
 
Stable isotopes can be employed to chemical and biochemical research in a couple of different 
ways.  In the first applica
tion, stable isotopes are employed as a mechanistic probe to help 
elucidate the detailed reaction mechanism.  For instance, stable C isotopes (
12
C and 
13
C) are 
often used to probe enzymatic reaction mechanisms related to central metabolism.  In one 
example
, Schmidt et al. determined the KIEs of the pyruvate dehydrogenase
-
catalyzed reactions 
by comparing the reaction rates of 
13
C
-
labeled and unlabeled substrates.
15
  
As mentioned in the 
previous section, to determine the intr
insic isotope effect, they assumed appropriate values for 
the values like forward and reverse commitment to catalysis based on past studies and stripped 
these factors away from the observed isotope effect.  The intrinsic isotope effect was then used to 
int
erpret the enzyme catalysis mechanism in terms of transition
-
state theory.  In the following 
reaction, 
 
H
3
CCOCO
2

3
CCOSCoA + CO
2
 
the KIE at C
-
2 position was 
12
k
/
13
k
 
= 1.0232 while the KIE at C
-
1 position was 1.051.  These 
results indicate that, as expec
ted, C
-
C bond cleavage is at least partially rate determining.  In 
addition, the smaller KIE value at the C
-
2 position than the C
-
1 position indicates that in the 
transition state, the loss of bond order at C
-
2 due to the cleavage of the C
-
C bond is partia
lly 
compensated by bond formation.
16
  
 
In another example, Waldrop et al analyzed kinetic isotope effects to probe the mechanism of 
a
spartate transcarbamoylase.
17
  
Specif
ically, using 
15
N isotope this group tested whether the 
reaction went through a cyanic acid or tetrahedral intermediate (Figure 5).
17
  
By comparing the 
measured intrinsic 
15
N KIE (1.0024
-
1.0027) with the measured KIEs from two model 
14
 
compounds (one utilizing a tetrahedral adduct mechanism (KIE
 
= 1.0028) and the other a 
cyanide acid mechanism (KIE = 1.0105)), Waldrop et al. deduced that the enzyme
-
catalyzed 
reaction mechanism utilizes a tetrahedral intermediate.  
 
 
 
Figure 5
:
 
 
Two proposed reaction mechanisms for the aspartate transcarbamoylase
 
catalyzed reaction. 
 
Scheme I is the tetrahedral intermediate mechanism and Scheme II is the 
cyanide intermediate mechanism.  
 
 
The second broad application employs stable isotopes as tracers.  In other words, stable isotopes 
are added into specific molec
ules to quantitatively determine the fluxes of a certain 
atom/metabolite in a particular pathway.  By analyzing the quantitative input and output of the 
15
 
labeled molecules, one can obtain a dynamic picture of the different fluxes of the metabolites in 
one o
r multiple pathways.  For example, by incorporating stable isotopes into key substrates such 
as glucose, one can monitor, quantify, and compare metabolic fluxes in the glycolic pathway in 
different tissues.  Using this approach, Fan et al.
18
 
were able to obtain a better understanding of 
lung cancer metabolomics.  In particular, by compar
ing the flux of Krebs cycle metabolites in 
cancerous and non
-
cancerous lung tissue, they determined that glycolysis was upregulated in 
cancerous tissue.  This discovery suggested new cancer treatment strategies
18
 
and led to the 
development biomarkers based on glycolytic and Krebs cycle intermediates to check for 
abnormal glucose metaboli
sm in potentially pre
-
cancerous tissue.
19
  
 
In addition,
 
stable isotopes can also be used as tracers in environmental studies or ecology, 
where they can be a powerful tool in tracking the migration of animals and the untangling of 
various food
-
web interactions.  For example, Hasson et al. analyzed stable N isot
opes to assess 
the diets of fish within the food
-
web structures of coastal sea areas, thereby allowing them to 
ascertain their migration pattern.
20
  
Another example is that by measuring and comparing the C 
isotope ratios in soils at different sites, one can determine whether the landscape was covered by 
grasses or forest trees in the past, as well as whether
 
the land was disturbed by human activities 
or left undisturbed.
21
  
 
Application of stable isotopes in biogeochemistry
 
The application of stable isotopes in biogeochemistry is in essence the same as in the fields of 
chemistry and biology, where stable iso
topes can be used both as tracers and as mechanistic 
probes.  Because of the uniqueness of biogeochemistry, however, the nomenclature and the 
processes are expressed slightly differently. 
 
16
 
The Rayleigh equation
 
The Rayleigh equations we use today originate
d from the Rayleigh distillation equation, which is 
described by: 
 
R/R
0
 
= (X
L
/X
L
0
)

-
1
                                                                                                                          
(9)
 
where R and R
0
 
are the isotope ratios of the reactant at a certain time and the reactant at the start 
of the reaction, respecti
vely, X
L
 
and X
L
0
 
are the concentration of the light isotope at a certain 

Rearrangement of Equation 6 leads to the following equation
22
: 
 

0
-

p/
s[f*lnf/(1
-
f)]                                                                                         
                       
(10)
 

0
 

p/s
 
is 

product
-

substrate
), 
and f is the fract
ion of the substrate remaining in a reaction.  
 
Strictly speaking, the Rayleigh equation describes the isotopic change in a chemically open 
system, assuming that the isotopic species removed at any instant is in equilibrium with those 
remaining in the syst
em.  Such ideal case rarely exists, however, and the Rayleigh equation is 
often used to approximate kinetic fractionations in closed systems.  
 
Although closed systems and open systems are quite different, the mathematical relationships 
derived from analys
is of open systems are often applied to closed systems.  Closed systems 
where the Rayleigh equation can be safely utilized are unidirectional reactions in which the 
atoms are transferred from one reservoir (substrate) to the second reservoir (product) unde
r 
constant isotopic equilibrium.  (For limitations to this approximation, see Kendall et al.
23
)  
17
 
Generally, most biological and geochemical s
ystems have normal kinetic isotope effects (i.e. the 
light isotope reacts more quickly than the heavy isotope).  In such systems, when the substrate is 
limited it gradually becomes enriched in the heavy isotope as the reaction proceeds.  
Concomitantly ther
e is a slow enrichment of the heavy isotopes in the final product over time 
because of the gradual enrichment of the heavy isotopes in the substrate pool (Figure 6).  In an 
open system where the substrate is infinite, the isotope ratio of the substrate doe
s not change 
over the time, and the fractionation between the substrate and product (instantaneous or 

can be expanded and can be applied to the closed systems 
where the substrate and product pools 
are constantly equilibrating.  Therefore, at all times in the closed system, the fractionation 
between the substrate and the 
instantaneous
 

 
 
18
 
 
Figure 6
:
  
Changes of isotope ratios of the substra
te, the instantaneous product, and the 
accumulated product in both open and closed systems. 
 
Modified from Kendall, 1998.
23
  
 
 
Conclusion
 
Sta
ble isotopes have been widely used by researches in biology, chemistry, and geochemistry.  
There are two major applications of stable isotopes.  One of the applications is to use them as 
tracers to track the fluxes of certain molecules in metabolic pathway
s.  In addition, stable 
isotopes can be used as a mechanistic probe by measuring the intrinsic kinetic isotope effect, 
which in turn can provide information about the reaction mechanism.  The field of 
19
 
biogeochemistry utilizes stable isotopes in similar app
lications, albeit presented in a different 
form because of the distinct expressions and terminology.
 
In this thesis, I employed stable isotopes as both tracers and mechanistic probes to study the 
microbial metabolism of two important gases.  In Chapter 2, 
a method to study and quantify the 
isotope fractionation related to hydrogenases (H
2
-
producing enzymes) is presented, which 
provides the basis for the studies in the next chapter.  In Chapter 3, an exhaustive study is 
presented where the isotope fractionat
ion related to various reactions catalyzed by different 
hydrogenases were quantified and compared.  The isotope effects determined in this study not 
only provided us with important mechanistic information, especially in the H
2
 
formation reaction, 
they also
 
enabled future studies that can utilize these fractionation factors as signatures to 
quantify the activity of hydrogenases in complex microbial communities.  In Chapter 4, isotope 
fractionation by a fungal N
2
O
-
producing enzyme (the nitric oxide reductase 
P450nor) is 
described.  The observed inverse isotope effect with the N

 
provided key information about the 
binding of the first NO molecule to the enzyme active site and the formation of the crucial 
intermediate in the enzyme mechanism.  In addition, I calculated the site preference (SP) values 
from the observed fractionatio
n patters, and these SP values can, in turn, be used as a tool to 
trace the source of N
2
O in the atmosphere.  
 
 
 
20
 
 
 
 
 
 
 
 
BIBLIOGRAPHY
 
 
 
21
 
BIBLIOGRAPHY
 
 
 
1.
 
Giauque, W. F.; Johnston, H. L., An isotope of oxygen, mass 18. 
Nature (London, U. 
K.) 
1929,
 
123
 
(Copyright (C) 2014 American Chemical Society (ACS). All Rights Reserved.), 318.
 
2.
 
Birge, R. T.; Menzel, D. H., The Relative Abundance of the Oxygen Isotopes, and the Basis of 
the Atomic Weight System. 
Physical Review 
1931,
 
37
 
(12), 1669
-
167
1.
 
3.
 
Urey, H. C.; Murphy, G. M.; Duncan, J. A., A HYDROGEN DISCHARGE TUBE FOR THE 
CONTINUOUS ULTRAVIOLET SPECTRUM. 
Review of Scientific Instruments 
1932,
 
3
 
(9), 497
-
498.
 
4.
 
Lewis, G. N.; Macdonald, R. T., Concentration of H2 Isotope. 
The Journal of Chemic
al Physics 
1933,
 
1
 
(6), 341
-
344.
 
5.
 
(a) Cremer, E.; Polanyi, M., Test of "tunnel" theory of heterogeneous catalysis on hydrogenation 
of styrene. 
Z. physik. Chem. 
1932,
 
B19
 
(Copyright (C) 2014 American Chemical Society (ACS). All 
Rights Reserved.), 443
-
50; 
(b) Eyring, H., The zero point energy and the separation of isotopes. 
Proc. 
Natl. Acad. Sci. U.S.A. 
1933,
 
19
 
(1), 78
-
81.
 
6.
 
Klein, F. S., Isotope Effects in Chemical Kinetics. 
Annual Review of Physical Chemistry 
1975,
 
26
 
(1), 191
-
210.
 
7.
 
Bigeleisen, J.; Wo
lfsberg, M., Theoretical and Experimental Aspects of Isotope Effects in 
Chemical Kinetics. In 
Advances in Chemical Physics
, John Wiley & Sons, Inc.: 2007; pp 15
-
76.
 
8.
 
Hornberger, G. M., New manuscript guidelines for the reporting of stable hydrogen, carbo
n, and 
oxygen isotope ratio data. 
Water Resources Research 
1995,
 
31
 
(12), 2895
-
2895.
 
9.
 
Hennig, C.; Oswald, R. B.; Schmatz, S., Secondary Kinetic Isotope Effect in Nucleophilic 

-

The Journal of Physical Chemistr
y A 
2006,
 
110
 
(9), 
3071
-
3079.
 
10.
 
Bigeleisen, J.; Lee, M. W.; Mandel, F., Equilibrium Isotope Effects. 
Annual Review of Physical 
Chemistry
 
1973,
 
24
 
(1), 407
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440.
 
11.
 
Kohen, A.; Klinman, J. P., Hydrogen tunneling in biology. 
Chemistry & Biology 
1999,
 
6
 
(7),
 
R191
-
R198.
 
12.
 
Cleland, W. W., Use of isotope effects to determine enzyme mechanisms. 
Journal of Labelled 
Compounds and Radiopharmaceuticals 
2007,
 
50
 
(11
-
12), 1006
-
1015.
 
13.
 
Northrop, D. B., The Expression of Isotope Effects on Enzyme
-
Catalyzed Reactions.
 
Annual 
Review of Biochemistry 
1981,
 
50
 
(1), 103
-
131.
 
14.
 
Northrop, D. B., Steady
-
state analysis of kinetic isotope effects in enzymic reactions. 
Biochemistry 
1975,
 
14
 
(12), 2644
-
2651.
 
22
 
15.
 
Melzer, E.; Schmidt, H. L., Carbon isotope effects on the pyruvate 
dehydrogenase reaction and 
their importance for relative carbon
-
13 depletion in lipids. 
Journal of Biological Chemistry 
1987,
 
262
 
(17), 8159
-
64.
 
16.
 
Klinman, J. P., The power of integrating kinetic isotope effects into the formalism of the 
Michaelis

Menten
 
equation. 
FEBS Journal 
2014,
 
281
 
(2), 489
-
497.
 
17.
 
Waldrop, G. L.; Urbauer, J. L.; Cleland, W. W., Nitrogen
-
15 isotope effects on nonenzymic and 
aspartate transcarbamylase catalyzed reactions of carbamyl phosphate. 
Journal of the American Chemical 
Society
 
1992,
 
114
 
(15), 5941
-
5945.
 
18.
 
Fan, T.; Lane, A.; Higashi, R.; Farag, M.; Gao, H.; Bousamra, M.; Miller, D., Altered regulation 
of metabolic pathways in human lung cancer discerned by 13C stable isotope
-
resolved metabolomics 
(SIRM). 
Molecular Cancer 
2009,
 
8
 
(1), 41.
 
19.
 
Vermeersch, K. A.; Styczynski, M. P., Applications of metabolomics in cancer research. 
Journal 
of 
Carcinogenesis
 
2013,
 
12
, 9.
 
20.
 
Hansson, S.; Hobbie, J. E.; Elmgren, R.; Larsson, U.; Fry, B.; Johansson, S., THE STABLE 
NITROGEN ISOTOPE RATI
O AS A MARKER OF FOOD
-
WEB INTERACTIONS AND FISH 
MIGRATION. 
Ecology 
1997,
 
78
 
(7), 2249
-
2257.
 
21.
 
Delègue, M.
-
A.; Fuhr, M.; Schwartz, D.; Mariotti, A.; Nasi, R., Recent origin of a large part of 
the forest cover in the Gabon coastal area based on stable carb
on isotope data. 
Oecologia 
2001,
 
129
 
(1), 
106
-
113.
 
22.
 
Scott, K. M.; Lu, X.; Cavanaugh, C. M.; Liu, J. S., Optimal methods for estimating kinetic 
isotope effects from different forms of the Rayleigh distillation equation 1. 
Geochimica et Cosmochimica 
Acta 
2004,
 
68
 
(3), 433
-
442.
 
23.
 
Kendall, C.; Caldwell, E. A., Chapter 2 
-
 
Fundamentals of Isotope Geochemistry. In 
Isotope 
Tracers in Catchment Hydrology
, McDonnell, C. K. J., Ed. Elsevier: Amsterdam, 1998; pp 51
-
86.
 
 
 
 
 
23
 
 
 
 
CHAPTER 2
 
U
SING
 
GAS
 
CHROMATOGRAPHY
-
I
SOTOPE
 
RATIO
 
MASS
 
SPECTROMETRY
 
TO
 
DETERMINE THE 
FRACTIONATION FACTOR FOR H
2
 
PRODUCTION BY 
HYDROGENASES
 
 
This chapter is modified from 
Rapid Communications in Mass Spectrometry
 
26(1): 
61
-
68 (2012) by 
Hui Yang, Hasand Gandhi, Liang Shi, Helen W. Kreuzer,
 
Nat
haniel E. Ostrom, and Eric L. Hegg
.
 
 
Hasand Gandhi and Nathaniel Ostrom designed the gas chromatography
-
isotope 
ratio mass spectrometry system and Hasand Gandhi additionally helped with the 
measurements.  Liang Shi provided the purified enzyme sample.  Hel
en W. 
Kreuzer provided key instructions, and Nathaniel Ostrom and Eric Hegg helped 
analyze data and directed the research.
24
 
ABSTRACT
 
Hydrogenases catalyze the reversible formation of H
2
, and they are key enzymes in the 
biological cycling of H
2
.  H isotopes 
have the potential to
 
be a very useful tool in quantifying 
hydrogen ion
 
trafficking in biological H
2
 
production processes, but there are several obstacles 
that have thus far limited the 
application
 
of this tool.  In this manuscript, we describe a new 
metho
d that overcomes some of these barriers and is specifically designed to measure isotopic 
fractionation during enzyme
-
catalyzed H
2
 
evolution.  A key feature of this technique is that 
purified hydrogenases are employed, allowing precise control over the reac
tion conditions and 
therefore a high level of precision.  In addition, a custom
-
designed high
-
throughput gas 
chromatography
-
isotope ratio mass spectrometer is employed to measure the isotope ratio of the 
H
2
.  Using our new approach, we determined that the 
fractionation factor 
for
 
H
2
 
production by 
the [NiFe]
-
hydrogenase from 
Desulfovibrio fructosovorans
 
is 0.273 ± 0.006.  This result 
indicates that, as expected, protons are highly favored over 
deuterium ions
 
during H
2
 
evolution.  
Potential applications of th
is newly developed method are discussed.
 
Key Words
 
 
Hydrogenase; H
2
 
production; fractionation factor; 
isotope ratio mass spectrometry
 
25
 
INTRODUCTION
 
 

consumption have led to conside
rable interest in alternative fuels.
[
1,2
]
  
H
2
 
is one attractive 
substitute 
due to its high energy content and the fact that it produces only water as the byproduct 
of combustion.
[
3
]
  
Currently H
2
 
is a critical chemical feedstock, but 96% of the H
2
 
produced 
today is derived from fossil fuels.
[
3
]
  
Conversely, microbially
-
produced H
2
 
is both renewable and 
carbon neutral, and the biological production of H
2
 
therefore has enormous potential to provide 
an environmentally friendly and sustainable source of energy.  
 
Although con
siderable progress has been made in elucidating the metabolic pathways 
involved in H
2
 
metabolism, many uncertainties remain.
[
4,5
]
  
One major impediment to improving 
our understanding of H
2
 
metabolism is our inability to define adequately the regulation of and 
the flux through key pathways involved in H
2
 
production.
[
6,7
]
  
In additi
on to producing H
2
, a 
number of microorganisms consume H
2
, and this biotic cycling plays a vital role in the anaerobic 
metabolism of many microbial communities.
[
8
-
10
]
  
Being able to quantify the biotic cycling of H
2
 
is critical to 
our understanding of H
2
 
metabolism and our ability to maximize biological H
2
 
production.
[
2,7,11
]
  
 
Hydrogenases are found in many
 
microbes where they catalyze the reversible reduction of 
protons to form H
2
:
 
 
2 H
+
 
+ 2 e
-
 

2
 
The two classes of hydrogenases that are particularly important in H
2
 
cycling are the [NiFe]
-
 
and 
[FeFe]
-
hydrogenases.
[
5
]
  
There is no evolutionary relationship between these two different 
26
 
cla
sses of hydrogenases, and as their name implies, they differ in the metal content at their active 
site.
[
12
-
14
]
  
O
ften organisms contain more than one hydrogenase, and it can therefore be difficult 
to ascertain the source and/or fate of biological H
2
 
in living systems.
[
15
]
  
Stable isotopes can be a 
powerful tool to trace fluxes through metabolic pathways,
[
8,16,17
]
 
and hydrogen isotopes have 
been used to study the abiotic cycling of H
2
.
[
18
]
  
Thus far, however, there are relatively few 
examples of using hydrogen isotopes to improve our knowledge of H
2
 
metabolism.
[
19
-
22
]
  
 
Fractionation factors for enzymes involved in H
2
 
metabolism can be used to help quantify 
the fluxes through different metabolic pathways.  There are two
 
strategies for measuring 

2
 
metabolism (defined as 

H2
/R
H2O
)
2
:  
in vivo
 
analysis in which one measures the isotope ratio of the H
2
 
produced or consumed by a whole 
organism, and 
in vitro
 
analysis in which the f
ractionation factor is determined directly from 
purified hydrogenases.  Recently the composite fractionation factor for H
2
 
uptake and H
2
-
H
2
O 
exchange was measured for five different organisms,
[
23
]
 
but the fractionation factor for H
2
 
production was not reported.  One of the major challenges in obtaining the 
in vivo
 
fractionation 
factor for H
2
 
production is that the isotope ratio of the H
2
 
formed is dependent on the isotope 
ratio of the substrate, i.e. the hydrogen ions in intracellular water.  Previously it was generally 
assumed that intracellular and extracellular water were effectively in c
omplete equilibrium, and 
that bulk water was therefore the source of the hydrogen ions in H
2
.  Our results, however, 
indicated that intracellular can be quite distinct from extracellular water due to the flux of 
hydrogen ions from organic substrates into i
ntracellular water during metabolic processing.
[
24
]
  
                                        
                  
 
2
 
The f
ractionation factor 
(

) 
is 
defined as 
R
A
/R
B
 
where R
A
 
and R
B
 
are the isotope ratio of the rare atom versus the 
abundant atom in the products and reactants, respectively
. 
 
Thus, in our studies of H
2
 

H2
/R
H2O
 
where R = 
2
H/
1
H.
 
27
 
Thus, to det
ermine the 
in vivo
 
fractionation factor for H
2
 
production, one must first measure the 
isotope ratio of intracellular water.
 
Determining the 
in vitro
 
fractionation factor of H
2
 
production presents many challenges as 
well, some of which are also encountered 
when calculating the 
in vivo
 
values.  First and foremost, 
the hydrogenase
-
catalyzed formation of H
2
 
is readily reversible.
[
13
]
  
Even under conditions in 
which H
2
 
formation is favored (e.g. very low partial pressures of H
2
 
or the presence of reducing 
agents), some H
2
 
uptake can be still be obser
ved.
[
25
-
28
]
  
Hydrogenases also catalyze a non
-
productive H
2
-
H
2
O exchange reaction in which the H atoms of H
2
 
exchange with H
2
O with no 
net formati
on or consumption of H
2
.  Because both H
2
 
consumption and H
2
-
H
2
O exchange of H
2
 
will alter the isotope ratio of H
2
 
in the headspace, these reactions will perturb the calculated 
fractionation factor for H
2
 
formation.  Thus, care must be taken to ascertain t
he extent to which 
these other reactions are occurring, and correct for them if needed.  A second challenge 
encountered when determining the 
in vitro
 
fractionation factors is that both [NiFe]
-
 
and [FeFe]
-
hydrogenases are inactivated by O
2
, necessitating an
aerobic purification and activity assays.  
 
In this paper, we describe an experimental protocol designed to measure the fractionation 
factors of H
2
 
production catalyzed by purified hydrogenases from microbial organisms.  As an 
important proof of concept, w
e calculated the fractionation factor of [NiFe]
-
hydrogenase from 
Desulfovibrio fructosovorans
.  To our knowledge, this is the first reported example of a fraction 
factor determined for a purified hydrogenase.
 
EXPERIMENTAL
 
H
2
 
E
volution 
A
ssay. 
 
28
 
Enzyme purifi
cation
.  The [NiFe]
-
hydrogenase from 
D. fructosovorans 
used in this experiment 
was purified from the native organism according to a modified literature procedure.
16
  
Briefly, 
D. 
fructosovorans
 
was obtained from ATCC (ATCC 49200; Manassas, VA, USA) and cultured 
anaerobically in SOS medium
[
29
]
 
at 37 
º
C for 72 h.  Following cell lysis and ultracentrifugation, 
the supernatant was loaded on a DEAE column (Bio
-
Rad, Hercules, CA, USA) equilibrated with 
Tris buffer A (10 mM Tris, pH 7.6), washed with Buffer A, and eluted with a gr
adient of 0
-
500 
mM NaCl.  The brown colored hydrogenase fraction eluted at about 70 mM NaCl, as confirmed 
by the hydrogen uptake activity assay.
[
29
]
  
The active fractions were pooled and subsequently 
loaded to a pre
-
packed cer
amic hydroxyapatite column (Bio
-
Rad, Hercules, CA, USA) 
equilibrated with 5 mM phosphate buffer saline (PBS).  The column was washed with 5 mM 
PBS and eluted with a stepwise gradient of 5 to 150 mM PBS.  Hydrogenase eluted at the end of 
the gradient.  The 
fractions with hydrogenase activity were pooled and filtered through a HiPrep 
16/60 Sephacryl S
-
200 HR gel filtration column with Tris buffer B (50 mM Tris, pH 8, and 150 
mM NaCl) by means of an AKTA Explorer fast protein liquid chromatography system (GE 
H
ealthcare, Piscataway, NJ, USA).  The active fractions were pooled, concentrated, and stored in 
Tris buffer B with 10% (vol/vol) glycerol at 
-
20 
º
C.  The purity of the isolated hydrogenase was 
confirmed by sodium dodecyl sulfate
-
polyacrylamide gel electrop
horesis. 
 
 
Sample preparation
.  In an anaerobic Coy chamber containing approximately 3% H
2
 
and 97% N
2
, 
50 mL of anaerobic H
2
 
evolution buffer (
10 mM methyl viologen, 80 mM Na
2
S
2
O
4
, 500 mM 
NaCl in 50 mL of 50 mM HEPES buffer, pH 7) 
was transferred to a 125 
mL borosilicate
-
glass 
serum vial (Wheaton Science Products, Millville, NJ, USA).  The vial was sealed with a gas
-
tight stopper (Bellco Glass, Vineland, NJ, USA) and crimped with an aluminum seal.  The vial 
was evacuated and refilled with Ar on a Schlenk li
ne several times to remove H
2
 
and N
2
 
from the 
29
 
headspace.  Purified hydrogenase in Ni
-
NTA buffer (100 mM Tris
-
HCl, pH 8, 100 mM 
imidazole, 200 mM NaCl, and 5% glycerol) was treated in an analogous manner.  
 
Measuring H
2
 
evolution
.  A 1 mL syringe was used t

vial containing the H
2
 
evolution buffer.  The reaction vial was then inverted and incubated at 
room temperature for 2 h, and the isotope ratio of the H
2
 
formed was measured via GC
-
isotope 
ratio mass spectrometry 
(GC
-
IRMS).  The amount of H
2
 
produced was quantified from the peak 
height (ion beam intensity at 
m
/
z
 
2) using a standard curve.  The concentration of H
2
 
in the 
headspace never exceeded 5%, thereby limiting nonproductive hydrogen ion exchange with 
water and
 
H
2
 
uptake.  To verify that the isotope ratio of the H
2
 
in the headspace was not 
significantly affected by the competing reactions, the ratio was monitored for 3 h, and no 

2
H over time were observed.
 
IRMS 
M
easurements
 
 
IRMS
 
Instrument setup
.  In this experiment, we use a new continuous flow design 
for measuring 

2
H of H
2
 

2
H
H2
) to enable determination of fractionation factors.  Our system maintains the 
high throughput capacity expected of continuous flow IRMS, while at the same time providing a 
high level of both precision (error of less than 3

low as 0.6 

mol gaseous H
2
 

7
 
depicts the 
continuous
-
flow gas chromatography isotope
-
ratio mass spectrometry (CF
-
GC
-
IRMS) system 

2
H
H2
.  The gas chromatograph (Hewlett
-
Packard # 5890 Series II, Palo 
Alto, CA, USA) is interfaced to an Elementar Isoprime stable isotope ratio mass spectrometer 
(Isoprime Limited, Cheadle, UK).  An ascarite trap is used to remove water before the sample 
g
as is introduced into a sample loop and then into the gas chromatograph and isotope ratio mass 
30
 
spectrometer.  A liquid N
2
 
trap was placed prior to the vacuum pump to facilitate water
-
removal 
between sample injections.  In the sample loading position (Figur
e 
7
A), the inlet system 
(consisting of the sample loop, tubing and injection port) is completely evacuated, and it is then 
isolated from the vacuum pump by closing stopcock 2.  The sample is injected via a gastight 
syringe into the inlet system on the gas 
chromatograph, and it immediately expands throughout 
the inlet system.  During this process, water is absorbed by the ascarite trap before a 2 mL gas 
sampling loop.  After approximately 10 s of equilibration, the 6
-
port, 2
-
position sampling valve 
(Valco In
struments, Houston, TX, USA) is switched to isolate the sample gases within the gas 
sampling loop (see Figure 
7
B).  The 4
-
port, 2
-
position sampling valve is then rotated to initiate 
He flow through the loop, pushing the sample gases onto the gas chromotogr
aph column (Figure 
7
C).  A 30 m × 0.53 mm o.d. molecular sieve 5 Å capillary column (Restek, Bellefonte, PA, 

separate H
2
 
from the rest of the sample gases.  The separated sample gases th
en enter the mass 
spectrometer, and the isotope ratio of the H
2
 
is determined and compared to the isotope ratio of 
the laboratory H
2
 
standard.  The total time of the sample analysis is approximately 400 s. 
 
31
 
 
Figure 
7
: 
 
Schematic representation of the GC
-
IRMS system designed for the stable isotopic 
analysis of gaseous H
2
. 
 
Arrows indicating directions of flow. See text for 
a 
detailed description.
 
32
 
Precision, accuracy and sensitivity of the instrument
.  The precision of our newly designed 
system was evaluat
ed by daily measurement of the Oztech standard (1 L gas cylinder standard 
purchased from Oztech, Waltham, MA, USA) injected into the inlet system (Table 
2
).  The 

comparing
 
2
H values of the working lab standards with the values determined 
by an independent laboratory (USGS, Denver, CO, USA) using a Thermo
-
Finnigan 252 mass 
spectrometer (Waltham, MA, USA) (Table 2).  The difference between the average values 
re
ported by the USGS (n = 9 for standard A, n = 6 for standard B) and in our laboratory is less 

of H
2
 
(Figure 
8

2
H values in agreement with the known i
sotope value of the tank 
standard (
-

(measured via injections of different amount of H
2

normal distribution, and the 95% confidence 
interval region was determined
.
[
30
]
  
The lower 
detection limit was determined as the injected H
2
 
amount that fell within the confidence intervals.  

2
 

 
 
Table 
2
:
 
 

2
H values of H
2
 
obtained for the Oztech H
2
 
reference gas. 
 
Date
 
Sample description
a
 
No. of 
samples
 

2

 
2010/5/3
 
syringe H
2
 
injection
 
4
 
-
65.2 ± 3.6
 
33
 
Table 2 (cont'd)
 
2010/5/17
 
 
syringe H
2 
injection
 
 
3
 
 
-
62.7 ± 2.2
 
2010/5/18
 
syringe H
2
 
injection
 
4
 
-
62.7 ± 0.4
 
mean ± SD
 
 
 
-
63.5 ± 2.0
 
Oztec
b
 
mean ± SD
 
 
 
-
62.7 ± 0.5
 
 
 
 
D = 0.8
 
a
The Oztech standard H
2
 
gas cylinder was used every day for adjustment of the MSU tank 
standard data. 
 
b

2
H values provided by the Oztech Trading Company in the Southern M
ethodist University 
Stable Isotope Laboratory. 
 
 
Table 
3
:
 
 

2
H values of MSU internal laboratory standards measured over three days as a 
function of the amount of H
2
 
injected. 
 
Date
 
Sample
 
Tank A
 
Tank B
 

2
 
 
2010
-
5
-
3
 
Syringe standard
a
 
-
412.1
 
-
339
.6
 
2010
-
5
-
3
 
Bottle standard
b
 
-
416.8
 
-
330.7
 
34
 
Table 3 (cont

d)
 
2010
-
5
-
17
 
 
Syringe standard
 
 
-
424.1
 
 
-
333.6
 
2010
-
5
-
17
 
Bottle standard
 
-
423.6
 
-
332.3
 
2010
-
5
-
18
 
Syringe standard
 
-
423.6
 
-
332.7
 
2010
-
5
-
18
 
Bottle standard
 
-
419.1
 
-
330.7
 
 
mean ± SD
 
-
419.9 ± 4.8
 
-
333.3 ± 3.3
 

2
 
 
2010
-
5
-
3
 
Syringe standard
 
-
419.0
 
-
336.3
 
2010
-
5
-
3
 
Bottle standard
 
-
413.8
 
-
328.7
 
2010
-
5
-
17
 
Syringe standard
 
-
422.4
 
-
329.1
 
2010
-
5
-
17
 
Bottle standard
 
-
423.6
 
-
332.3
 
2010
-
5
-
18
 
Syringe standard
 
-
423.6
 
-
331.7
 
2010
-
5
-
18
 
Bottle standard
 
-
422.1
 
-
3
28.2
 
 
mean ± SD
 
-
420.8 ± 3.8
 
-
331.0 ± 3.1
 

2
 
 
2010
-
5
-
3
 
Syringe standard
 
-
420.3
 
-
333.9
 
2010
-
5
-
3
 
Bottle standard
 
-
426.2
 
-
335.5
 
35
 
Table 3 (cont

d)
 
2010
-
5
-
18
 
 
Syringe standard
 
 
-
423.6
 
 
-
332.4
 
 
mean ± SD
 
-
423.3 ± 2.9
 
-
333.9 ± 1.6
 

2
 
 
2
010
-
5
-
3
 
Syringe standard
 
-
424.4
 
-
333.4
 
2010
-
5
-
3
 
Bottle standard
 
-
422.5
 
-
331.0
 
2010
-
5
-
18
 
Syringe standard
 
-
424.4
 
-
332.6
 
 
 
mean ± SD
 
-
423.8 ± 1.1
 
-
332.3 ± 1.2
 
 
All MSU data mean ± 
SD
 
-
421.4 ± 3.9
 
-
332.5 ± 2.8
 
 
 
USGS
c
 
mean ± SD
 
-
424.3 
± 1.6
 
-
330.0 
± 1.8
 
 
 

 

 
a
Syringe standards were H
2
 
gas standards introduced as pure gas samples into the gas 
chromatograph
-
isotope ratio mass spectrometer using a gas
-
tight syringe.
 
b
Bottle standards were prepared by adding H
2
 
into a 13 mL serum bottle filled wit
h Ar at 
atmospheric pressure; 1 mL of the H
2
/Ar mixed gas was then injected using a gas
-
tight syringe.
 
c
Samples of MSU tank standards were sent to the USGS laboratory for an independent analysis. 
A total of 9 analyses were conducted at the USGS over three 
days. 
 
36
 
 
Figure 
8
:
 
 
Determination of the 

2
H by the GC
-
IRMS system
.  In the 


the precisi
on becomes unacceptable.
 
 

2
H
2
O
  
 

2
H of H
2

2
H
H2O
) of the reactant solution was determined using a Los Gatos Research 

2
H
H2O
 
was 
-

 
Da
ta Analysis
  
 

2
H.  
To correct the raw isotope ratio value, a 1 L stainless steel cylinder 
containing pure H
2
 
with a known 
2
H/
1
H ratio (
-

37
 
Ocean Water)) was acquired from Oztech Trading Corporation (Dall
as, TX, USA).  This 
reference gas and two other laboratory working standards (
-

-

VSMOW as determined by the USGS using a dual
-
inlet isotope ratio mass spectrometer) were 
used on a daily basis to calibrate the system.  The software, 
MassLynx (Elementar Americas Inc., 
Mt. Laurel, NJ, USA), performs an internal correction for baseline drift and the contribution of 
H
3
+
 
to the mass 3 detector. 
 
Standardization and notation
.  All results are reported with respect to VSMOW, whose absolute 
r
atio of 
2
H/
1
H is 155.76 (±0.05) × 10
-
6
.  The relative ratio of a sample with respect to VSMOW is 
commonly given by the relationship: 
 

2
H = (R
sample
/R
VSMOW
 

 

 
where R = 
2
H/
1
H.
 
RESULTS AND DISCUSSION
 
In this manuscript, we present a GC
-
IRMS met
hod to measure isotopic fractionation during 
hydrogenase
-
catalyzed H
2
 
production.  The headspace gas from a series of reactions was 
measured to determine the fractionation factor of 
[NiFe]
-
hydrogenase from 
D. fructosovorans.
  
Like all catalysts, [NiFe]
-
hyd
rogenases can catalyze both the forward and the reverse reaction, as 
well as a H
2
-
H
2
O exchange reaction.  All of these reactions can occur even when the H
2
 
partial 
pressure is low.
[
27,28
]
  
Thus, the isotope ratio of the H
2
 
in the headspace may be influenced by 
reactions other than the evolution reaction.  To limit the influence of the competing reactions, an 
excess amount of a strong reductant (80 mM Na
2
S
2
O
4
) was used in 
the experiments to maintain a 
large thermodynamic driving force for H
2
 
production and to saturate the enzyme to ensure 
38
 
maximal velocity.  In addition, the enzyme concentration was adjusted such that the 
concentration of H
2
 
in the headspace never rose above
 
5%, and the reactions were performed for 

2
 
uptake and H
2
-
H
2
O exchange.  
Importantly, time course experiments were performed for each data set, and 
our results 
2
H
H2
 
value re
mained constant
 
(Table 3).  Because the rates of the 
competing reactions increase as H
2
 
accumulates in the headspace, the observed isotope ratio of 
the headspace H
2
 
would most likely change over time if either of the competing reactions were 
influencing th
e observed 

2
H
H2
.
3
  
The fact that the isotope ratio remained constant 
is consistent 
with isotope fractionation from an infinitely large reservoir of water
, and it provides a strong 
indication that the competing reactions are not significant under our reaction conditi
on.  
 
 
Table 
4
:
 
 
Isotope ratio of H
2
 
produced by 
Desulfovibrio fructosovorans 
hydrogenase
.
 
Name
 
Time (h)
 
Ht (nA)
 

2
H 

 
Fractionation facto
r 
 
(a)
a
 
 
2009
-
7
-
21
 
Df Sample A
b
 
1
 
4.03
 
-
728.5
 
0.275
 
 
 
2
 
5.36
 
-
735.1
 
0.283
 
 
 
3
 
6.31
 
-
735.6
 
0.284
 
 
                                        
                  
 
3
 
Both the H
2
-
consumption and the nonp
roductive exchange reactions are first order with respect to [H
2
].  
These two 
reactions 
will perturb the observed 

2
H
H2
 
value 
except in the unlikely event that 
the 
combined 
fractionation of the 
competing reactions
 
exactly matches the fractionation of H
2
 
pr
oduction.
 
39
 
Table 4 (cont'd)
 
Df Sample B
 
 
1
 
 
3.80
 
 
-
730.4
 
 
0.275
 
 
 
2
 
4.88
 
-
731.8
 
0.275
 
 
 
3
 
6.90
 
-
736.6
 
0.286
 
 
2009
-
8
-
6
 
Df Sample C
 
1
 
3.58
 
-
762.7
 
0.254
 
 
 
2
 
5.14
 
-
757.6
 
0.264
 
 
 
3
 
6.00
 
-
759.3
 
0.262
 
 
Df Sample D
 
1
 
3.46
 
-
763.1
 
0.254
 
 
 
2
 
5.08
 
-
757.4
 
0.264
 
 
 
3
 
5.67
 
-
751.7
 
0.254
 
 
2010
-
11
-
23
 
Df Sample E
 
1
 
3.30
 
-
722.0
 
0.295
 
 
 
2
 
4.22
 
-
733.6
 
0.280
 
 
 
3
 
6.12
 
-
743.9
 
0.274
 
 
2010
-
12
-
1
 
Df Sample F
 
1
 
5.70
 
-
737.3
 
0.280
 
 
40
 
Ta
ble 4 (cont'd)
 
 
 
2
 
 
6.37
 
 
-
735.8
 
 
0.274
 
 
 
3
 
7.11
 
-
737.8
 
0.281
 
 
 
4
 
7.85
 
-
744.7
 
0.272
 
 
mean ± SD
 
 
 
-
742.4 
± 12.6
 
0.273 ± 0.006
 
 
a
The fractionation factor is defined as R
H2
/R
H2O
, taking into account the H
2
 
dissolved in the 
solution. 
 
b
Df refers to 
D. fru
ctosovorans 
[NiFe]
-
hydrogenase. A to F indicate six different reaction 
preparations. 
 
 
Calculations for determining the fractionation factor for H
2
 
evolution
 
The fractionation factor for H
2
 
evolution is defined as 
 

H2evolution
 
= R
H2(T)
/R
H2O
 
 
 
 
 
 
 
 
 
(1)
 
where 
R
H2(T)
 
and R
H2O
 
is the ratio of deuterium to hydrogen in the total H
2
 
product and in the 
reactant (i.e. water), respectively.  Thus, to calculate the fractionation factor of the [NiFe]
-
hydrogenase, we must first
 
know the isotope ratio of the H
2
 
produced.  Because H
2
 
can dissolve 
and fractionate between the gas and liquid phases, however, the fractionation factor determined 
using only the headspace H
2
 
must be corrected to account for this partitioning.
 
41
 
This partit
ioning can be described using the mass balance equation
[
31
]
 
R
H2(T)
 
= X
H2(gas)
R
H2(gas)
 
+ X
H2(sol)
R
H2(sol)
 
 
 
 
 
 
 
(2)
 
where R
H2(T)
, R
H2(gas)
, and R
H2(sol)
 
are the isotope ratios of the total H
2
 
produced, the H
2
 
in th
e 
headspace, and the H
2
 
in the solution, and X
H2(gas)
 
and X
H2(sol)
 
represent the molar fraction of H
2
 
in the headspace and in the solution, respectively.  R
H2(sol)
, however, is related to R
H2(gas)
 
by the 

H2equal
 

H2equal
 
= R
H2(gas)
/R
H2(sol)
 
= 1.067),
[
32
]
 
and equation 2 therefore 
becomes: 
 
R
H2(T)
 
= X
H2(gas)
R
H2(gas)
 
+ X
H2(sol)
R
H2(gas)

H2equal
 
 
 
 
 
 
(3)
 
This equation describin
g a two
-
component mixture simplifies to:
 
R
H2(T)
 
= X
H2(gas)
R
H2(gas)
 
+ (1 
-
 
X
H2(gas)
)R
H2(gas)

H2equall
 
 
 
 
 
(4)
 
Because the temperature and pressure remain essential constant during the course of the reaction,
 
the molar fraction of H
2
 
in the gas phase (X
H2(g
as)
) can be approximated as 
 
X
H2(gas)
 
= V
H2(gas)
/(V
H2(gas)
+V
H2(sol)
)
 
 
 
 
 
 
 
 
(5)
 
where V
H2(gas)
 
and V
H2(sol)
 
represent the volume of the H
2
 
in headspace and in solution, 
respectively.  The amount of H
2
 
dissolved in solution can be calculated using 
the follo
wing 
equation
 
V
H2(sol)
 
= V
solution
 

 
 
 
 
 
 
 
 
 
(6)
 


10
-
3
 
at 25 ºC and 1 atm), V
solution
 
is the volume of the 
reaction solution (50 mL), and V
H2(sol)
 
is the volume of H
2
 
dissolved in the solution.
[
33
]
  
Thus, 
using the known constants and measurements from these experiments, 
R
H2(T)
 
can b
e quantified.  
42
 
The fractionation factor of the hydrogenase
-
catalyzed H
2
 

H2evolution
) reaction can 
therefore be readily calculated using equation 1.
 
The fractionation factor for the production of H
2
 
catalyzed the [NiFe]
-
hydrogenase from 
D. 
fructosovran
 
was calculated multiple times, and the results are shown in Tab
le 3.  As noted 
above, these values do not vary over time.  The average value of the fractionation factor is 
0.27±0.01, indicating that protons react significantly faster than deuterium ions during H
2
 
formation.  Interestingly, our calculated fractionation
 
factor of 0.
27 is in general agreement with 

[
34
]
  
This net 
fractionation factor, however, is the result of multiple H
2
 
fractionation steps in the cell, and the 
percentage of H
2
 
formed by hydrogenase versus nitrogenase in these cultures is not known.  Thus, 
any direct comparisons between our 
fractionation factor determined using purified enzymes and 
the net fractionation factor calculated for cyanobacterial cultures must be made with caution.
 
Potential Applications 
 
The findings presented here illustrate the potential for this method to provid
e precise and robust 
fractionation factors for H
2
 
evolving enzymes.  These values can, in turn, expand our knowledge 
and understanding of microbial H
2
 
metabolism.  In one potential application, knowledge of the 
fractionation factors can be used to ascertai
n the source of H
2
.  Because fractionation factors are 

H2evolution

step in the reaction pathway, the observed fractionation factor is specific to the reaction 
coordin
ate profile.  While [NiFe]
-
 
and [FeFe]
-
hydrogenases obviously have dissimilar active 

H2evolution
 
values 
due to slight differences in H
-
bonding and hydrogen ion transport.  Ther
efore, despite the fact 
43
 
that all hydrogenases produce H
2
 
from protons and electrons, hydrogenase enzymes will 

total
.  These differences in the fractionation factors will be 
manifested in the isotope ratio of the H
2
 
pro
duced, thus providing each enzyme with its own 
unique signature.  In an organism that contains two hydrogenases (either naturally or through 
genetic engineering), the relative activity of each hydrogenase can therefore be determined by 
measuring the isotop
e ratio of H
2
.  
 
In a second application, the fractionation factors can be used to test for proton channeling.  
Although it is well established that the addition of organic substrates can increase H
2
 
production 
in some organisms,
[
35
-
37
]
 
the precise mechanism by which this occurs is not entirely clear.  
Do the 
organic substrates merely provide an additional source of electrons to the general cellular pool, 
or are the two processes more directly coupled?  For example, in 
Shewanella oneidensis
 
the 
addition of certain organic substrates increases H
2
 
producti
on even when electron acceptors are 
present,
[
15
]
 
suggesting that some channeling of electrons and protons may be occurring.  A 
comparison of the 
in vitro
 
and 
in vivo
 
hydrogenase fractionation factors can be used to test this 
phenomenon.  If channeling is occurring, the source of protons will not be
 
the bulk intracellular 
water, and this will result in different apparent 
in vivo
 
and 
in vitro
 
hydrogenase fractionation 
factors. 
 
Finally, knowledge of the fractionation factors can also aid in environmental reconstruction.  
Many organisms consume H
2
 
as a
n energy supply, and H
2
 
is consequently a source of cellular 
hydrogen atoms for lipid biosynthesis in these organisms.
[
19
]
  
The fractionation factor of H
2
 
uptake is therefore needed to interpret fully the isotope ratios of sedimentary lipids during 
geochemical reconstruction.
 
44
 
The instrument design described here is compatible
 
with a wide variety of applications.  
Because pre
-
treatment of the sample is not required,
[
38
]
 
the design is well suited for high 
throughpu
t measurement of enzymatic reactions.  Furthermore, it is possible to configure the 
system such that a sample can be loaded directly from the side arm of a culture bottle or assay 
vial into the evacuated sample loop, thereby avoiding the potential for frac
tionation or leakage 
caused by syringe injections.
[
39
]
 
 
CONCLUSIONS
 
The fractionation factors determined for 
hydrogenases can greatly aid in our understanding of H
2
 
metabolism.  The method described in this paper is both fast and accurate, and it provides a 
rigorous strategy for quantifying the fractionation factor for H
2
 
evolution (

H2evolution
).  As a proof 
of concept, we determined 
H2evolution
 
for the [NiFe]
-
hydrogenase from 
D. fructosovorans
.  The 
large discrimination observed in this reaction (

H2evolution
 
= 0.27) indicates that hydrogen ions 
react much faster than deuterium ion
s during H
2
 
evolution.  This fractionation factor can be used 
as a signature for the 
D. fructosovorans
 
[NiFe]
-
hydrogenase and aid in the analysis of H
2
 
metabolizing pathways.  Significantly, because this method is completely generic, it can be 
applied to a
 
variety of other enzymes involved in H
2
 
metabolism.  
 
Acknowledgements
 
We thank James J. Moran from Pacific Northwest National Lab (PNNL) for helpful discussions.  
Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.
S. 
Department of Energy under Contract No. DE
-
AC05
-
76RL01830.  Financial support was 
provided by the U.S. Department of Energy (DOE), Office of Biological and Environmental 
45
 

(
#1053432) is also gratefully acknowledged.
 
 
 
 
46
 
 
 
 
 
 
 
 
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47
 
 
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L. Cournac, G. Guedeney, G. Peltier, P. M. Vignais. Sustained photoevolution of 
molecular hydrogen in a mutant of 
Synechocystis sp.
 
strain PCC 6803 deficient in the type 
I NADPH
-
dehydrogenase complex. 
J. Bacteriol.
 
2004
, 
186
, 1737.
 
50
 
[38].
 
A. L. Sessions. Isotope
-
ratio detection for gas chromatography. 
J. Sep. Sci.
 
2006
, 
29
, 1946.
 
[39].
 

18
O of 
dissolved and gaseous dioxygen via gas chromatography
-
isotope ratio mass spectrometry. 
Environ. Sci. Technol.
 
2000
, 
34
, 2337. 
 
 
 
51
 
 
 
CHAPTER 3
 
ISOTOPIC FRACTIONATI
ON ASSOCIATED WITH [NiFe]
-
 
AND [FeFe]
-
HYDROGENASES
 
 
 
Hasand Gandhi helped with the gas chromatography
-
isotope ratio mass 
spectrometry analyses.  Adam J. Cornish provided constructs for four enzyme 
samples.  Liang Shi and John Peters kindly provided purifie
d [NiFe]
-
hydrogenases 
from 
Desulfivibrio fructosovorans
 
and 
Thiocapsa roseopersicina
, respectively.  
James J. Moran and Helen W. Kreuzer helped with the analyses and discussion.  
Nathaniel Ostrom and Eric Hegg helped analyze data and directed the research.
 
 
52
 
ABSTRACT
 
RATIONALE
: Hydrogenases catalyze the reversible formation of H
2
 
from electrons and 
protons with high efficiency.  Understanding the relationships between H
2
 
production, H
2
 
uptake, 
and H
2
-
H
2
O exchange can provide insight into the metabolism of m
icrobial communities in 
which H
2
 
is an essential component in energy cycling.  
 
METHODS
: In this manuscript, we used stable H isotopes (
1
H and 
2
H) to probe the isotope 
effects associated with three [FeFe]
-
hydrogenases and three [NiFe]
-
hydrogenases.  
 
RESUL
TS
: All six hydrogenases displayed fractionation factors for H
2
 
formation that were 
significantly less than 1, producing H
2
 
that was severely depleted in 
2
H relative to the substrate, 
water.  Consistent with differences in their active site structure, the 
fra
ctionation factors for each 
class appear to cluster, with the three [NiFe]
-

-
0.40) generally having 
smaller values than the three [FeFe]
-

-
0.55).  We also obtained isotopic 
fractionation factors associated with H
2
 
uptake and H
2
-
H
2
O exchange under conditions similar to 
those utilized for H
2
 
production, providing us with a more complete picture of the three reactions 
catalyzed by hydrogenases.  
 
CONCLUSIONS
: The fractionation factors determined in our studies can be u
sed as signatures 
for different hydrogenases to probe their activity under different growth conditions and to 
ascertain which hydrogenases are most responsible for H
2
 
production and/or uptake in complex 
microbial communities.  
 
Key Words
 
53
 
[FeFe]
-
hydrogenase
; [NiFe]
-
hydrogenase; fractionation factor; kinetic isotope effect (KIE); 
hydrogen isotopes; isotope ratio mass spectrometry (IRMS)
 
INTRODUCTION
 
Microbial H
2
 
production is of great interest to chemists, biologists, and biogeochemists.  
Because H
2
 
has the h
ighest gravimetric energy density (
-
242 kJ mol
-
1
)
[
1
]
 
of any fuel and can be 
produced from renewable sources both chemically and biochemically, it is often consid
ered as a 
potential alternative energy source.  In addition, H
2
 
is a key metabolite produced and/or 
consumed by many bacteria and algae.  In natural ecosystems H
2
 
is often transferred between 
different microbial communities during syntrophic growth,
[
2
-
5
]
 
and the flux of H
2
 
through these 
systems can therefore provide important information about the interactions of these microbial 
communities.  
 
Two of the most importa
nt classes of enzymes involved in microbial H
2
 
production and 
consumption are [FeFe]
-
 
and [NiFe]
-
hydrogeneases.
[
6
, 
7
]
  
These enzymes do not share any 
evolutionary relationships, and as their names imply, they differ significantly in their active site 
structures, with [FeFe]
-
hydrogenases containing a diiron center and [NiFe]
-
hydrogenases 
harboring a NiFe heterodinucle
ar center (Figure 
9
).
[
8
, 
9
]
  
In [FeFe]
-
hydrogenases, only the distal 
iron atom changes oxidation state in
 
the observed intermediates of the catalytic cycle,
[
10
]
 
and the 
nitrogen atom of the unusual bridging dithiomethylamine ligand is proposed to play an integral 
role in
 
proton transfer.
[
6
, 
11
]
  
Conversely, in [NiFe]
-
hydrogenases all of the redox chemistry 
happens at the Ni atom, a
nd one of the cysteine ligands is proposed to participate in the acid
-
base 
chemistry.
[
6
, 
12
]
  
In addition, [Fe
Fe]
-
hydrogenases tend to be more active in H
2
 
generation than 
[NiFe]
-
hydrogenases, with an 
in vitro
 
rate for the [FeFe]
-
hydrogenases of up to 8.2 mmol 
54
 
H
2
·
min
-
1
·
mg protein
-
1
 
(~ 10
4
 
molecules of H
2
 
per second per molecule of enzyme).
[
13
]
  
Thus, 
despite the fact that both [FeFe]
-
 
and [NiFe]
-
hydrogenases catalyze the reversible reduction of 
protons to H
2
, they utilize different reaction mechanisms.
[
8
]
  
 
 
 
Figure 
9
:
  
Structures of the active sites in [NiFe]
-
 
and [FeFe]
-
hydrogenases
 
(modified from 
Lubitz et al.
[
6
]
).  
 
 
 
Hydrogenases are capable of catalyzing t
he three following reactions: (1) H
2
 
production 
(2H
+
 
+ 2e
-
 

2
),
[
7
]
 
(2) H
2
 
consumption via the heterolytic cleavage of H
2
 
(H
2
 

+
 
+ H
-
 

2H
+
 
+ 2e
-
),
[
14
]
 
and (3) H
2
-
H
2
O exchange.
[
15
]
  
In the exchange reaction, H
2
 
binds to the active site 
and is heterolytically cleaved to a proton and a metal
-
bound hydride 
as in the H
2
 
consumption 
reaction,
[
16
]
 
but then the proton exchanges with the surrounding water before recombining with 
the metal
-
bound hydride to regenerate H
2
.  Depending on the physiological conditions, more 
than one of these reactions may be occurring in the cell simultaneously. 
 
Due to these 
complexities, it is therefore advantageous
 
when studying microbial H
2
 
production
 
to isolate and 
analyze both H
2
 
consumption and H
2
-
H
2
O exchange as well.
 
55
 
Hydrogen isotopes are uniquely suited to be a powerful and non
-
invasive tool for 
investig
ating microbial H
2
 
metabolism.  Not only is the movement of hydrogen ions an essential 
component of most of the key steps of the reaction pathway, but the isotope effects tend to be 
large due to the substantial relative difference in mass between 
1
H and 
2
H
.  Consequently, 
hydrogen isotopes have been widely utilized over the years to interrogate hydrogenase function 
and mechanism.  For example, experiments studying H
2
-
H
2
O exchange have used hydrogen 
isotopes as a tracer to track the proton
-
hydride recombinat
ion process
[
17
, 
18
]
 
and as a mechanistic 
probe to confirm that H
2
 
is split by hydrogenases via heterolysis.
[
19
]
  
 
The different activ
e site structures and reaction mechanisms of [NiFe]
-
 
and [FeFe]
-
hydrogenases will inevitably lead to distinct reaction profiles.  Not only might this result in 
unique isotopic preferences for these two classes of enzymes, we propose that even enzymes 
withi
n the same class can have different isotopic preferences due to slight differences in substrate 
binding, proton transfer, hydrogen bonding, etc. that result in slight perturbations in the reaction 
profile.  Thus, in theory each hydrogenase can have its own
 
unique isotopic signature for both H
2
 
production and consumption.  These isotopic signatures can be measured via isotope ratio mass 
spectrometry (IRMS)
§
 

product
/R
substrate
, where 
R
product
 
and R
substrat
e
 
are the isotope ratio of the product and substrate, respectively.  If these 
isotopic signatures can be precisely measured, then it may be possible to identify the source of 
hydrogenase activity in a complex community.
[
20
]
 
 
                                        
                  
 
§
 
Abbreviations: 
CpFeFe
,
 
Clostridium pasteur
i
an
um
 
[FeFe]
-
hydrogenase
; 
CrFeFe
,
 
Chlamydomonas reinhardtii
 
[FeFe]
-
hydrogenase
; 
DfNiFe
,
 
Desulfovibrio fructosovorans
 
[NiFe]
-
hydrogenas
e; GC
-
IRMS, gas chromatography
-
isotope ratio mass spectrometry;
 
IRMS
, isotope ratio mass spectrometry; KIE, kinetic isotope effect;
 
SoFeFe
,
 
Shewanella oneidensis
 
[FeFe]
-
hydrogenase
; 
SoNiFe
, 
Shewanella oneidensis
 
[NiFe]
-
hydrogenase
; 
TrNiFe
, 
Thiocapsa 
roseopersicina
 
[NiFe]
-
hydrogenase
; V
SM
OW, 
Vienna Standard Mean Ocean Water
.  
 
56
 
In this manuscript we report fractionation factors associated with H
2
 
production, H
2
 
uptake, an
d H
2
-
H
2
O exchange for 6 different hydrogenases under similar conditions.  To our 
knowledge, this is the first manuscript that compares a variety of fractionation factors among 
different hydrogenases as well as different hydrogenase reactions.  
 
 
EXPERIMENT
AL
 
Enzyme preparation.
  
 
The [FeFe]
-
hydrogenase from 
Clostridium pasteurianum 
(CpFeFe), the [FeFe]
-
hydrogenase 
CrHydA1 from 
Chlamydomonas reinhardtii 
(CrFeFe), and both the [FeFe]
-
 
and [NiFe]
-
hydrogenases from 
Shewanella oneidensis
 
MR
-
1 (SoFeFe and SoNiFe,
 
respectively) were 
cloned into a modified pAC
-
BAD vector, overexpressed in 
S. oneidensis
 
MR
-
1, and purified via 
Ni
-
NTA (Ni
-
nitrilotriacetic acid, Qiagen, Valencia, CA) affinity chromatography as described by 
Cornish et al.
[
21
]
  
Briefly, the coding sequence of the hydrogenases were amplified by PCR and 
inserted into the NcoI/SacI sites of the plasmid pAC
-
BAD which contains a kanamycin 
resistance marker and an 
L
-
arabinose inducible promoter.  The resu
lting plasmids were 
transformed into a 
S. oneidensis 
strain lacking the native [FeFe]
-
 
and [NiFe]
-
hydrogenases 
(

) but retaining the native maturation proteins required to assemble active 
hydrogenase.  Following expression and purification,
[
21
]
 
the active hydrogenase fractions were 
pooled, concentrated, and stored in Tris buffer (100 mM Tris
-
HCl, pH 8.0, 200 mM NaCl, 10 
mM sodium dithionite) with 10% (vol/vol) glycerol at 
-
20 ºC.  Sodium do
decyl sulfate
-
polyacrylamide gel electrophoresis of the purified hydrogenases showed that approximately 80% 
57
 
of the total protein in the lane was present in a single band corresponding to the hydrogenase.  
The 
Desulfivibrio fructosovorans
 
hydrogenase (DfNiF
e), kindly provided to us by Liang Shi 
(Pacific Northwest National Lab, Richland, WA), was purified from the native organism as 
described previously.
[
22
]
  
The [NiFe]
-
hydrogenase from 
Thiocapsa roseopersicina
 
(TrNiFe
) was 
provided by John Peters (Montana State University, Bozeman, MT).
[
23
, 
24
]
 
H
2
 
evolution assay
. 
 
H
2
 
evo
lution activity assays were performed using the procedure published in Yang et al.
[
25
]
  
Briefly, sodium di
thionite (100 mM final concentration) and methyl viologen (20 mM final 
concentration) were dissolved in 100 mM Tris buffer (pH 8.0) in an anaerobic Coy chamber 
(filled with 2
-
4% H
2
 
balanced with N
2
), sealed in a 125 mL serum vial (Wheaton Science 
Products,
 
Millville, NJ, USA), and then degassed on a Schlenk line to remove excess H
2
.  
Following the anaerobic addition of 100 µL of the appropriate hydrogenase (~ 0.4 mg/mL) using 
a gas
-
tight syringe, the concentration and isotope ratio of H
2
 
in the headspace we
re monitored 
and analyzed via continuous flow gas chromatography
-
isotope ratio mass spectrometry (GC
-
IRMS)
[
2
5
]
 
over the course of 3
-
4 h.  The amount of H
2
 
in the headspace was quantified from the 
peak height (ion beam intensity at 
m/z
 
= 2) using a standard curve, and the changes of the isotope 
ratios over time were fit to a Rayleigh equation to obtain the frac
tionation factors.  Because 
hydrogenases can catalyze both H
2
 
uptake and H
2
-
H
2
O exchange in addition to H
2
 
formation, the 
concentration of H
2
 
in the headspace was monitored and maintained below 5%.  In addition, 
excess reductant was used to drive the react
ion towards H
2
 
evolution and mitigate the effects of 
the competing reactions.  
 
58
 
 
The raw data (Table 
5
) was subjected to a one
-
sided t test to determine the outliers
[
26
]
 
in 
which the mean values of 6 samples for CrFeFe, and mean values of 8 samples for CpFeFe were 
used.  Two observations were ruled as outliers based on one
-
tailed 5% significance levels.  In 
ad
dition, the data for H
2
-
H
2
O exchange (Figure 
10
)
 
were also checked for outliers based on 5% 
confidence level by using a one
-
sided t test.  In total, our analyses include data from 5 biological 
replicates for CrFeFe, 7 biological replicates for CpFeFe, 4 bi
ological replicates for both SoFeFe 
and SoNiFe, 6 biological replicates for DfNiFe, and 2 biological replicates for TrNiFe.  
 
 
Table 
5
: 
 

2
H data for production of H
2
 
by different hydrogenases.
 
Name
 
Time 
(h)
 
H
2
a
 

mol/mL
)
 

2

 
Average
 
CrFeFe hydrogenase
 
9/30/2010
 
Cr Sample 
1
 
1
 
1.65
 
-
546.0
 
 
 
3
 
2.36
 
-
584.3
 
-
565.1
 
10/1/2010
 
Cr Sample 
2
 
1
 
2.09
 
-
603.5
 
 
 
2
 
2.59
 
-
614.7
 
 
 
 
3
 
2.95
 
-
621.1
 
-
613.1
 
11/22/2010
 
Cr Sample 
3
 
1
 
1.56
 
-
543.2
 
 
59
 
Table 5 (cont

d)
 
 
3
 
1.74
 
-
563.5
 
-
553.4
 
Cr Sample 
4
 
1
 
1.18
 
-
567.7
 
 
 
2
 
1.12
 
-
570.6
 
 
 
3
 
1.42
 
-
523.7
 
 
 
4
 
1.20
 
-
578.1
 
 
 
5
 
1.12
 
-
579.6
 
-
563.9
 
11/23/2010
 
Cr Sample 
5
 
1
 
1.89
 
-
548.9
 
-
548.9
 
mean ± SD
 
 
 
 
 
 
-
568.9 ± 25.7
 
CpFeFe hydrogenase
 
6/9/2011
 
Cp Sample 
1
 
2
 
1.55
 
-
450.4
 
 
 
3
 
2.93
 
-
505.5
 
 
 
 
4
 
1.21
 
-
540.1
 
-
498.7
 
8/4/2011
 
Cp Sample 
2
 
1
 
1.72
 
-
498.4
 
 
 
2
 
3.16
 
-
514.2
 
-
506.3
 
Cp Sample 
3
 
1
 
2.14
 
-
492.9
 
 
 
2
 
2.50
 
-
495.1
 
-
494.0
 
8/5/2011
 
Cp Sample 
4
 
1
 
1.4
1
 
-
465.6
 
 
60
 
Table 5 (con

d)
 
 
2
 
1.86
 
-
471.0
 
-
468.
3
 
Cp Sample 
5
 
1
 
0.89
 
-
349.6
 
 
 
2
 
1.03
 
-
445.8
 
-
397.7
 
Cp Sample 
6
 
1
 
0.60
 
-
427.1
 
 
 
2
 
1.40
 
-
461.3
 
 
 
 
3
 
2.03
 
-
472.1
 
-
453.5
 
2/6/2012
 
Cp Sample 
7
 
1
 
0.16
 
-
518.7
 
 
 
2
 
0.16
 
-
519.0
 
 
 
3
 
0.30
 
-
520.9
 
-
519.5
 
mean ±
 
SD
 
 
 
 
 
 
-
476.9 ± 41.5
 
SoFeFe hydrogenase
 
9/30/2010
 
SoFe Sample 
1
b
 
1
 
0.35
 
-
632.4
 
 
 
2
 
0.41
 
-
639.9
 
 
 
3
 
0.45
 
-
639.1
 
-
637.1
 
8/5/2011
 
SoFe Sample 
2
 
1
 
2.62
 
-
645.5
 
-
645.5
 
11/7/2012
 
SoFe Sample 
3
 
1
 
0.55
 
-
588.67
 
 
 
2
 
0.56
 
-
590.26
 
 
61
 
Table 5 (cont

d)
 
 
3
 
0.6
8
 
-
601.28
 
-
593.40
 
SoFe Sample 
4
 
1
 
0.55
 
-
590.64
 
 
 
2
 
0.57
 
-
594.52
 
-
592.58
 
mean ± SD
 
 
 
 
 
 
-
617.2 ± 
28.1
 
SoNiFe hydrogenase
 
8/5/2011
 
SoNi Sample 
1
 
3
 
0.78
 
-
633.3
 
-
633.3
 
SoNi Sample 
2
 
3
 
0.81
 
-
630.7
 
-
630.7
 
11/7/2012
 
SoNi Sample 
3
 
1
 
0.32
 
-
617.57
 
 
 
2
 
0.34
 
-
620.19
 
 
 
3
 
0.39
 
-
629.13
 
-
622.30
 
SoNi Sample 
4
 
1
 
0.32
 
-
621.32
 
 
 
2
 
0.34
 
-
618.91
 
-
620.11
 
mean ± SD
 
 
 
 
 
 
-
626.6 ± 
6.39
 
DfNiFe hydrogenase
 
7/21/2009
 
Df Sample 
1
 
1
 
0.63
 
-
728.5
 
 
 
 
 
2
 
0.84
 
-
735.1
 
 
 
 
 
3
 
1.00
 
-
735.6
 
-
733.1
 
Df Sample 
2
 
1
 
0.59
 
-
730.4
 
 
 
62
 
Ta
ble 5 (cont

d)
 
 
 
2
 
0.76
 
-
731.8
 
 
 
 
 
3
 
1.09
 
-
736.6
 
-
732.9
 
8/6/2009
 
Df Sample 
3
 
1
 
0.55
 
-
762.7
 
 
 
 
 
2
 
0.81
 
-
757.6
 
 
 
 
 
3
 
0.95
 
-
759.3
 
-
759.9
 
Df Sample 
4
 
1
 
0.53
 
-
763.1
 
 
 
 
 
2
 
0.80
 
-
757.4
 
 
 
 
 
3
 
0.89
 
-
751.7
 
-
757.4
 
11/23/2010
 
Df Sample 
5
 
1
 
0.51
 
-
722
 
 
 
2
 
0.
66
 
-
733.6
 
 
 
 
3
 
0.96
 
-
743.9
 
-
733.2
 
12/1/2010
 
Df Sample 
6
 
1
 
0.90
 
-
737.3
 
 
 
2
 
1.01
 
-
735.8
 
 
 
3
 
1.13
 
-
737.8
 
 
 
 
4
 
1.25
 
-
744.7
 
-
738.9
 
mean ± SD
 
 
 
 
 
 
-
742.6 ± 
12.7 
 
TrNiFe hydrogenase
 
9/9/2009
 
63
 
Table 5 (cont

d)
 
TrNiFe Sample 1
 
1
 
0.22
 
-
723.69
 
 
 
2
 
0.25
 
-
72
5.46
 
 
 
3
 
0.28
 
-
724.32
 
 
 
4
 
0.31
 
-
723.05
 
-
724.13
 
TrNiFe Sample 2
 
1
 
0.12
 
-
725.13
 
 
 
2
 
0.14
 
-
726.37
 
 
 
3
 
0.16
 
-
726.56
 
 
 
4
 
0.18
 
-
726.86
 
-
726.23
 
mean ± SD
 
 
 
 
 
 
-
725.18 ± 1.05
 
 
a
The H
2
 
concentrations were calculated by comparing the peak heights of the samp
les generated 
via GC
-
IRMS to those of the standards with known amounts of H
2
.  Concentrations have not 
been corrected for sampling loss (~1% per injection).
 
 
64
 
 
Figure 
1
0
:
  

2
H measurements of H
2
-
H
2
O exchange reactions catalyzed by different 
hydrogenases over the course of 180 min.  
 
 
H
2
 
uptake assay
. 
 
A
 
hydrogen uptake assay was performed similarly to the H
2
 
formation assay except that H
2
 
and 
the oxidant benzyl viologen we
re present instead of a reductant.  The redox potential of benzyl 
viologen is approximately 
-
0.370 V vs the standard hydrogen electrode,
[
27
]
 
providing the driving 
force for H
2
 
oxid
ation.  Briefly, 50 mL of uptake buffer (100 mM Tris
-
HCl, pH 8.0, 200 mM 
NaCl, 10 mM benzyl viologen, 5% glycerol) were sealed in a 125 mL serum vial in an anaerobic 
Coy chamber with approximately 5% H
2
 
in the headspace (the maximum concentration of H
2
 
pre
sent in the headspace during H
2
 
evolution experiments).  To initiate the reaction, a 1 mL 

65
 
then inverted and shaken at room temperature.  The reaction was allowed to proceed for 3
-
4 h, 
and 1 mL 
of the headspace was analyzed every ~60 min.  The amount and isotope ratio of the H
2
 
that remained after the various time points were analyzed via GC
-
IRMS as described above for 
the H
2
 
evolution assay.
 
H
2
-
H
2
O exchange assay
. 
 
A 1 mL syringe was used to inj

serum vial containing approximately 5% H
2
 
in the headspace (to simulate the maximum H
2
 
present in the headspace during the H
2
 
evolution experiments) and 50 mL of reaction buffer (100 
mM Tris
-
HCl, pH
 
8.0, 200 mM NaCl, 5% glycerol).  The mixture also contained one enzyme 
equivalent of the oxidant benzyl viologen to poise the enzyme for H
2
 
binding and exchange.  The 
reaction vial was then mixed, inverted, and incubated at room temperature while shaking.
  
The 
isotope ratio of the H
2
 
was measured via GC
-
IRMS for up to 50 h, and the amount of H
2
 
remaining in the headspace was quantified from the peak height (ion beam intensity at m/z 2) 
using a standard curve.  
 
Standardization and notation.
  
 
All results a
re reported with respect to VSMOW (Vienna Standard Mean Ocean Water), whose 
absolute ratio of 
2
H/
1
H is 155.76 (±0.05) × 10
-
6
.
[
28
]
  
The relative ratio of a sample with respect to 
VSMOW is commonly given by the relationship: 
 

2
H = (R
sample
/R
VSMOW
 

 

 
where R = 
2
H/
1
H.
 
66
 

 

product
/R
reactant, 
 
where R
product
 
and R
reactant
 
are isotope ratios of the product and reactant, respectively.  For H
2
 

H2
/R
H2O
, while for H
2
 
upta

H2O
/R
H2
.  Under our experimental conditions 
the water was in vast excess in these reactions, and R
H2O
 
was therefore essentially constant 
during the course of our reactions.
 
The kinetic isotope effect (KIE) is a related term that describes the diffe
rence in rate between the 
two isotopes:
 
KIE = 
k
light
/
k
heavy
 
where 
k
light
 
and 
k
heavy
 
are the rate constants for the light (
1
H) and heavy (
2
H) isotopes, 
respectively.
[
29
]
  

another:
 

product
/R
reactant
 
= (
k
light
/
k
heavy
)
-
1
 
= KIE
-
1
 
RESULTS
 
Isotopic fractionation associated with H
2
 
evolution
 
Figure 
11
 
and Table 
6
 
report the isotope ratios of 
H
2
 
produced by six different hydrogenases as 

2
H = 
-

these experiments can be considered an infinite reservoir, R
H2
 

67
 
the isotope ratio of
 
the H
2
 
produced can therefore be used to characterize the fractionation 
patterns by different hydrogenases.  From the figure, it can be seen that CrFeFe and CpFeFe 
produced H
2
 

2
H values of approximately 
-

-

, 
DfNiFe and TrNiFe both generated H
2
 

2
H values of 
roughly 
-

-

S. 
oneidensis
 
produced H
2
 
with isotope ratios that were between these two extremes
, and within 
error indistinguishable from each other (p = 0.55), with SoFeFe generating H
2
 

2
H value 
of approximately 
-

2
 
with an isotope ratio of roughly 
-

 
 
 
Figure 
11
:
  
Isotope ratio of H
2
 
produced by different hydro
genases
.  For each enzyme, at 
least 3 samples were analyzed to calculate the average and standard deviation values.  
 
68
 
Table 
6
:
  

2
 
production catalyzed by different [NiFe]
-
 
an
d [FeFe]
-
hydrogenases
.  Values are expressed as 
the mean ± SD (standard deviation).  
 
 

 

 

 

 

 

 




 
-




 
-




 
-




 
-




 
-




 
-




 




 


 


 





 




 




 



 



 


 


 



 



 



 



 
 
To ensure that the results obtained were not significantly altered by the competing 
pathways (i.e H
2
 
consumption or H
2
-
H
2
O ex
change), experimental conditions were set such that 
excess reductant was used to drive the reaction toward H
2
 
production and the headspace H
2
 
concentration was kept below 5% to minimize the back reaction.  In addition, analyses were 
carried out to determin
e if there was a correlation between the rate of the reaction and the extent 
of H
2
 
production.  The rationale was that H
2
 
consumption (which would alter the apparent H
2
 
production rate) is strongly dependent on the partial pressure of H
2
.  Therefore, if H
2
 
consumption is occurring to a significant extent in our reaction, the apparent H
2
 
production rate 
should slow in a predictable manner over time.  Figure 
1
2
, however, indicated that there was no 
obvious correlation between H
2
 
production rate and the extent
 
of reaction, suggesting that H
2
 
69
 
consumption is not significant under our reaction conditions.  Nevertheless, the variations 
observed in the isotope ratio of H
2
 
produced by some of the hydrogenases, particularly CrFeFe 
and CpFeFe (the two hydrogenases that
 
were the most active in H
2
 
production) suggested that 
competing reactions might be occurring despite our best efforts to minimize them.
 
 
 
Figure 
12
:
 
 
Measured H
2
 
production rate over the course of the analyses
.  No obvious 
correlation was observed betwee
n the apparent rate of H
2
 
production and the extent of the 
reaction.
 
70
 

calculated for H
2
 

2
H of the H
2
 
produced by each enzyme over time 
(Figu
re 
1
3
, Table 
5

2
H 
value for H
2
 
production should be observed in the absence of competing reactions.  If, however, 
either H
2
 
uptake or H
2
-
H
2
O exchange (which have distinct fractionatio
n factors) where occurring 
to a large extent under our H
2
 
production reaction conditions, then the rates of these competing 
reactions would increase over time as the concentration of H
2
 
in the headspace increased.  This 
would, in turn, alter the isotope ra
tio of the headspace H
2
 
in a time
-
dependent manner that 
reflected the increased importance of the isotope effects associated with the secondary 
reaction(s).  For most of the hydrogenases used in t

2
H with 
reaction time was observed (Figure 
1
3
), suggesting that these competing reactions were not a 
significant factor under our experimental conditions.  
 
71
 
 
Figure 
1
3
:
  

2
H of the 
H
2
 
produced in H
2
 
evolution 
reaction tested
.  In different graphs, different symbol represents different samples tested.  
 
 
In the case of CpFeFe, however, the H
2
 
produced became more depleted in 
2
H over time 
in approximately half of the experiments (Table
 
5
, Figure 
1
3

2
H in 
the CpFeFe reaction could be explained if H
2
-
H
2
O exchange drove the accumulated H
2
 
toward a 

2
H = 
-


2
H = 
-

formation for calculation) between H
2
 
and H
2
O.
[
30
]
  
Alternatively, H
2
 
uptake may have been occurring, in which case our data indicates this reaction 
72
 
would have a preference for 
2
H (i.e. an inverse KIE).  To address
 
these issues, we performed 
experiments to measure independently both H
2
 
uptake and H
2
-
H
2
O exchange for each of the 
hydrogenases.  
 
Isotopic fractionation associated with H
2
 
consumption
 
To quantify the isotope effect associated with enzymatic H
2
 
consumptio
n, H
2
 
with a precisely 
defined isotope ratio was incubated with each of the hydrogenases in the presence of a strong 
oxidant, benzyl viologen.  The reaction conditions mimicked those used in the H
2
 
production 
assay, including maintenance of the headspace H
2
 
concentration below 5%, with the exception 
that an electron acceptor was used instead of an electron donor.  Both the amount and the isotope 
ratio of H
2
 
remaining in the headspace at each time point were used to fit the Rayleigh 
equation
[
31
, 
32
]
 
to calculate the fractionation factors associated with each enzyme (Figure 
1
4
).  
 


















 
+







 
 
 
 
 
 
 
 
(1)
 

 
= 

/1000 + 1
 
 
 
 
 
 
 
 
 
 
 
(2)
 
where 







 
is the isotope ratio of H
2
 
at different time points, 
f
 
is the fraction of H
2
 
remaining 

factor related to the uptake reaction.  
 
73
 
 
Figure 
14
:
  
Relationships between isotope ratio of headspace H
2
 

2
) and the fraction of 
H
2
 
consumed (
f
) for 4 different hydrogenases tested.
  
According to the Rayleigh equation, the 
slope of the line is the enri

 
 
As shown in Table 
7
, the fractionation factors calculated for H
2
 
consumption associated 
with each of the four different hydrogenases tested are very similar to each other.  This 
seemingly surprising result can be easily explained if H
2
 
diffusion from the headspace into 
solution is the rate
-
limiting step in the reaction, thereby masking the true fractionation factor for 
H
2
 
consumption associated with each hydrogenase.  
 
 
74
 
Table 
7
: 
 

sured for H
2
 
consumption (i.e. oxidation) catalyzed by different [NiFe]
-
 
and [FeFe]
-
hydrogenases. 
 
Samples were monitored for approximately 3 hours as described in the Experimental section.  
Values are expressed as the mean ± SD (standard deviation).  
 
 
Cr
FeFe
 
CpFeFe
 
SoFeFe
 
SoNiFe
 

 
-
143.10 ± 39.36
 
-
170.78 ± 36.32
 
-
116.17 ± 12.72
 
-
122.88 ± 14.13
 

 
0.86 ± 0.04
 
0.83 ± 0.04
 
0.88 ± 0.01
 
0.88 ± 0.01
 
KIE 

 
1.17 ± 0.05
 
1.21 ± 0.05
 
1.13 ± 0.02
 
1.14 ± 0.02
 
 
Isotopic fractionation associated with H
2
-
H
2
O exchange reaction
 
The H
2
-
H
2
O exch
ange isotopic fractionation factor was quantified to assess whether this reaction 
might be contributing to the isotopic variations observed in the H
2
 
production experiments.  Thus, 
H
2
-
H
2
O exchange reactions were established using experimental conditions si
milar to the H
2
 
production reactions only without the addition of reductant.  Furthermore, the isotopic 
compositions of the H
2
 
and H
2

2
H
-
H
2
 

-


2
H
-
H
2
O = 
-

2
H
-
H
2
 

2
H
-
H
2
 
at 
equilibrium is calculated to be 
-

[
30
]
).  Although m
inor variations at initial time 

2
H of H
2
 
were evident (perhaps due to a small degree of isotope fractionation during 
75
 
introduction of the H
2
 
gas into the headspace), the isotopic ratio headspace H
2
 
did not vary 
substantially during 3 h measuremen
ts (Figure 
10
), suggesting that H
2
-
H
2
O exchange is not 
occurring to a great extent under our reaction conditions.  
 
DISCUSSION
 
Three important observations can be made from the H
2
 
production results presented in Figure 
11
 
and Table 
6
.  First, when producin
g H
2
, all of the hydrogenases have fractionation factors much 
less than one and therefore produce H
2
 
that is severely depleted in 
2
H relatively to H
2
O.  This 
indicates that, as expected, protons (
1
H) react to form H
2
 
faster than deuterons (
2
H) react, 
consi
stent with deuterons forming stronger and less labile hydrogen bonds.
 
The second important observation is that the data seem to partition into two separate 
clusters (Figure 
1
5
, p = 0.01), with the [FeFe]
-

and p
roducing H
2
 
that is typically more enriched in 
2
H relative to the H
2
 
produced by the [NiFe]
-
hydrogenases.  (It should be noted that while the SoFeFe and SoNiFe enzymes appear to follow 
these general trends, the H
2
 
produced from these two enzymes is indisti
nguishable based on the 
t
-
test (p = 0.55), making any definitive distinction of H
2
 
production from these two enzymes 
tenuous.)  To our knowledge, this is the first report of fractionation factors for purified [FeFe]
-


 
0.55).  However, our calculated fractionation factors for the [NiFe]
-


 
0.4
0) agree well with other published results in which fractionation 
factors of 0.3
-
0.5 can be calculated.
[
18
, 
33
, 
34
]
  
Together, these observations are consistent with our 
hypothesis that [FeFe]
-
 
and [NiFe]
-
hydrogenases generally produce isotopically distinct H
2
 
due 
to differences in their active site structures.  
 
76
 
 
Figure
 
15
:
  

2
H of the H
2
 
produced by [FeFe]
-
hydrogenases 
versus [NiFe]
-
hydrogenases. 
 
The H
2
 
produced by the [FeFe]
-
hydrogenases is statistically 
different from the H
2
 
produced by the [NiFe]
-
hydrogenases (p = 0.01).  The shaded area 
in the 
box plot represents the 25
-
75 percentile for that data set, the line represents the median, the dot 
indicates the average, and the error bars represent highest/lowest values observed.
 
 
Third, even within the same class, different hydrogenases can po
ssess different 
fractionation factors and produce H
2
 
with different isotope ratios.  In fact, the variation within an 
enzyme class can be larger than the variation between classes.  This result was unexpected 
77
 
because the amino acids involved in the proton 
transport pathway are predicted to be conserved 
within each class.
[
21
, 
35
]
  
The relatively large variation of
 

differences in the extent to which the true isotope effect is masked by other processes such as 
product release (i.e. the release of H
2
 
after enzyme catalysis), proton transfer, or the extent to 
which each proton transfe
r step is reversible under our reaction conditions (i.e. the commitment 
to catalysis).  Consistent with this idea, Hexter et al. noted that different hydrogenases within the 
same class can have substantially different activation enthalpies,
[
36
]
 
which could affect the 
reversibility of in
dividual steps.  Our current data do not allow us to discriminate between these 
different possibilities. 
 
One question that remains to be resolved is why the isotope ratio of the H
2
 
produced by 
some of the CpFeFe samples became further depleted in 
2
H over 
time.  This phenomenon could 
be explained if H
2
 
uptake (for which the rate relative to production would increase over time as 
the concentration of H
2
 
in the headspace increased) were occurring to a significant extent and if 
1
H
-
2
H were oxidized faster than 
1
H
2
 
(i.e. if there were an inverse KIE).  The data in Table 
7
, 
however, indicates that CpFeFe exhibits a normal KIE during H
2
 
oxidation, indicating a 
preference for the lighter isotope.  This finding is consistent with previous studies of metal
-
hydrogen bi

2
-
H
2
) complexes which display normal equilibrium 
isotope effects of 1.217 to 1.685.
[
37
]
  
In addition, because both of the M
-
H
2
 

H2

M
 
and d
M

*
H2
)
[
38
, 
39
]
 
will inevitably lead to a weakening of the H
-
H bond in the transition state, a 
normal KIE can also be anticipated for H
2
 
binding and splitting.  Thus, H
2
 
uptake cannot explain 
the
 
results obtained for CpFeFe.
 
78
 
In theory, H
2
-
H
2

2
H in the accumulated 
H
2
 
observed over time during the CpFeFe H
2
 
production assay.  This reaction is well 
documented in a number of different hydrogenases,
[
17
, 
18
, 
40
, 
41
]
 

2
H 
value for H
2
 

2

-

[
42
]
) of 
-

[
30
]
.  If 
CpFeFe is very efficient at catalyzing this exchange reaction, it could explain the trends we 
observe in approximately half of the CpFeFe H
2
 
production reactions.  Figur
e 
10
, however, 
indicates that no significant H
2
-
H
2
O exchange is occurring during any of our experiments, all of 
which utilize relatively low concentrations of natural abundance H
2
 
for short periods of time.  
Thus, neither H
2
 
uptake/oxidation nor H
2
-
H
2
O exc

2
H in 
the accumulated H
2
 
produced by CpFeFe, and further experiments will be needed to explain this 
observation.
 
The kinetic isotope effects that we have systematically measured in purified [FeFe]
-
 
and 
[NiFe]
-
hydroge
nases can provide key insights into microbial H
2
 
metabolism.  For example, the 
relative difference between the H
2
 
produced by the 
S. oneidensis 
[FeFe]
-
 
and [NiFe]
-

in vivo
 
studies.
[
20
]
  
Interestingly, the 

2
H
 
values of H
2
 
measured in vivo by Kreuzer et al. 
(
-

-

-
 
and 
[NiFe]
-
hydrogenases, respectively) 
[
20
]
 
differ from these current 
in vitro
 
studies by approximately 

[
42
, 
43
]
  
Isotopic gradients between intracellular and extracellular water are known to exist in certain 
circumstances,
[
44
-
46
]
which could alter the isotope ratio of H
2
 
produced 
in vivo
 
relative to what 
would be expected based on the growth medium water.  However, both the [FeFe]
-
 
and [NiFe]
-
hydrogenase in 
S. oneidensis
 
are located in t
he periplasm, and it is not clear whether a sufficient 
isotopic gradient is maintained across the outer membrane to account for the observed difference 
79
 

2
H of H
2
 
between the in vivo and in vitro experiments.  Nevertheless, the nearly identical 
relative 
differences in 

2
H between 
S. oneidensis 
[FeFe]
-
 
and [NiFe]
-
hydrogenases observed in 
the two studies is intriguing.  
 
Kreuzer et al.
[
20
]
 
used a combination of 
in vivo
 
transcription data and hydrogen isotope 
ratios to ascertain when the [FeFe]
-
 
and [NiFe]
-
hydrogenases in wild
-
type 
S. oneidensis
 
were 
expressed and active
.  Transcription did not always correlate with activity, highlighting the 
importance of developing techniques to measure enzyme activity directly when analyzing H
2
 
production in complex biological communities.  The variation in fractionation factors and ki
netic 
isotope effects we observe for [FeFe]
-
 
and [NiFe]
-

2
H of the H
2
 
produced can be one important tool.
 
In conclusion, we determined the fractionation factors and the kinetic isotope effects for 
both H
2
 
production and H
2
 
up
take for three purified [FeFe]
-
hydrogenases and three purified 
[NiFe]
-
hydrogenases.  The large normal isotope effects observed for H
2
 
production for all six 
hydrogenases indicates that, as expected, protons react faster than deuterons to form H
2
.  In 
addit
ion, the calculated fractionation factors seem to cluster, with the [FeFe]
-
hydrogenases 
generally having larger fractionation factors than [NiFe]
-
hydrogenases, consistent with our 
hypothesis that variations in the active site will contribute to different i
sotopic preferences.  
Interestingly, differences within the same class can be just as large or larger than the differences 
between classes, indicating that other factors are also affecting the reaction coordinates and the 
isotopic preferences.  Importantly
, the isotopic fractionation factors reported here provide a basis 
for distinguishing which enzymes are active in a complex microbial community where H
2
 
is used 
as an energy carrier between different species.  
 
80
 
Acknowledgements
 
Financial support was provid
ed by the U.S. Department of Energy (DOE), Office of Biological 

Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. 
Department of E
nergy under Contract No. DE
-
AC05
-
&6RL01830.  Support from NSF 
(#1053432) is also gratefully acknowledged.  
 
 
 
81
 
 
 
 
 
 
 
 
 
 
 
APPENDIX
 
 
82
 

2 
after equilibration with H
2
O
 
The fractionation factor for the H
2
-
H
2
O exchange is defined as 
 

H2O
/R
H2
 
where R
H2O
 
and R
H2
 
are the isotope ratios for the H
2
O and H
2
, respectively.  
 
T
he isotope ratio of H
2
O (R
H2O

water, where 
 

2
O = 
-

 
R
H2O
 

2
O/1000 + 1)*R
H2O(standard)
 
The R
H2O(standard)
 
is known to be 155.76*10
-
6
 
for the H isotope, and R
H2O
 
is calculated to be 
1.46*10
-
6
.  
 
According to Horibe et al.
[
31
]

2
-
H
2
O exchange is 
defined as 
 

2
 
+ 2.060*10
9
/T
4
 
+ 0.180*10
15
/T
6
 
where T is the temperature in K
elvin.  
 

 
R
H2
 
= R
H2O

-
6
/3.83 = 3.81*10
-
5
 
83
 

2
 
value following equilibration with Michigan tap water to be 
-

 
 
 
 
 
 
84
 
 
 
 
 
 
 
 
 
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85
 
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oxidizing hydrogenase 
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Biochim. Biophys. Acta 
1982
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I. Sumner, G.A. Voth. Proton transport pathways in [NiFe]
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hydrogenase. 
J. Phys. Chem. B 
2012
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6]
 
S.V. Hexter, F. Grey, T. Happe, V. Climent, F.A. Armstrong. Electrocatalytic mechanism of 
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Proc. Natl. Acad. Sci. U.S.A. 
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L. Torres, 
R. Gelabert, M. Moreno, J.M. Lluch. Nuclear dynamics discrete variable representation 
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2
 

2
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H
2
)L
n
 

theoretical way to account for anharmonicity. 
J. Phys. Chem. A 
2000
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[38]
 
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Acc. Chem. Res. 
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P.J. Hay. Ab initio theoretical studies of a novel tungsten dihydrogen complex. 
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[40]
 
P.M. Vignais. H/D exchange reactions and mechanistic aspects of the hydrogenases. 
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Chem. Rev. 
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bound 
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Coplen. Distribution of oxygen
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H.W. Kreuzer
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Martin, M.J. Lott, J.R. Ehleringer, E.L. Hegg. Metabolic processes account for the 
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-
Martin, J.R. Ehleringer, E.L. Hegg. Oxygen isotopes indicate most intracellular 
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2012
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7
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89
 
 
 
 
 
 
CHAPTER 4
 
ISOTOPIC FRACTIONATOIN BY A FUNGAL P450 NITRIC 
OXIDE REDUCTASEDURING THE PRODUCTION OF N
2
O
 
 
This chapter is modified from the 
Environmental Science & Technology
 
48(18): 
10707
-
10715 (2014) by Hui Yang, Hasand Gan
dhi, Nathaniel E. Ostrom, Eric L. 
Hegg
 
Hasand Gandhi performed isotope ratio analyses of the N
2
O molecules.  Nathaniel 
Ostrom and Eric Hegg helped analyze the data and directed the research.
 
 
 
90
 
Abstract
 
Nitrous oxide (N
2
O) is a potent greenhouse gas with a 
100
-
year global warming potential 
approximately 300 times that of CO
2
.  Because microbes account for over 75% of the N
2
O 
released in the U.S., understanding the biochemical processes by which N
2
O is produced is 
critical to our efforts to mitigate climate c
hange.  In the current study, we used gas 
chromatography
-
isotope ra
tio mass spectrometry (GC
-

15

18

15
N

, 

15
N

 
of N
2
O generated by purified fungal nitric oxide reductase (P450nor) from 
Histoplasma capsulatum
.
  
The isotope values were 
used to calculate site preference (SP) values 
(difference
 

15

2
O), 
enrichment factors 

  
Both oxygen and N

 
displayed normal isotope effects 
during enzymatic NO reduction
 

-

-
12


15
N of N

 
and N

) and N

 
exhibited
 

15
N

 
is co
nsistent with reversible binding 
of the first NO in the P450nor reaction mechanism.  In contrast to the constant SP observed 
during NO reduction in microbial cultures, the site preference measured for purified 
H. 
capsulatum
 
P450nor was not constant, increa

reaction.  This indicates that SP for microbial cultures can vary depending on the growth 
conditions, which may complicate source tracing during microbial denitrification. 
 
Keywords: enrichment factor, fungal
 
NO reductase, isotopomer, kinetic isotope effect, nitrous 
oxide, P450nor, site preference
 
 
91
 
Introduction
 
The nitrogen cycle is a fundamental biogeochemical process that is crucial to all living 
organisms.  Two especially important components of the nitroge
n cycle are nitrification (a 
process in which ammonia is oxidized into nitrate), and denitrification (the reduction of nitrate 
and nitrite to N
2
).  Nitrous oxide (N
2
O), a potent greenhouse gas that is also involved in ozone 
layer destruction, is produced b
oth as an intermediate and as a side product during microbial 
nitrification and denitrification.
48
  
With the rise of agricultural activities and the associa
ted use of 
fertilizers, N
2
O levels have risen to approximately 325 ppb (19% higher than pre
-
industrial 
levels),
49
 
and they are currently increasing at a rate of 0.25% per
 
year.
50
  
Because microbial 
denitrification is one of the major sources of biologically generated N
2
O,
51
 
it is imperative that 
we obtain a better understanding of the specific enzymatic steps involved in N
2
O generation 
during denitrification.  
 
Quan
tifying the magnitude of isotopic discrimination at natural abundance levels during 
biochemical reactions provides a non
-
invasive and physiological way to probe biological N
2
O 
production.
52
  
Evaluation of isotopic fractionation during denitrification, however, is generally 

composition between nitrate and N
2
 
in either microbial cultures
53
 
or controlled field settings.
54
  
Ultimately these studies describe the fractionation
 
for the entire denitrification sequence (NO
3
-
 

2
) or a series of steps rather than the fractionation occurring at an individual step (e.g. NO 

2

15
N at the central (N

) and the 
terminal (N

) positions) and O isotope values can be helpful
, they are often inconclusive.  For 
example, microbial denitrification generates N
2
O depleted in 
15
N relative to nitrate between 
-

and 
-

2
O production via nitrification of 
-

92
 
to 
-

53a
, 
55
  
This difference in fractionation patterns has been used to classify N
2
O production 
in soils as coming either from de
nitrification or nitrification.  The observed fractionation, 

and masks the true fractionation occurring at a specific enzymatic transformation.
56
  
Further, 
exchange of O between water and N
2
O during its production is common and can
 
alter the isotope 
signature of a specific production pathway.
57
  
Thus, the specific microbial source(s) of N
2
O 

15

18
O often cannot be quantified.  
 

15
N of the central and terminal N atom 
in the asymmetric N
2
O molecule, has emerged as an additional tool by which N
2
O biosynthesis 
can be characte

15

18
O values is that it is 
independent of the isotopic composition of the inorganic substrates,
58
 
and thus fa
r no evidence of 
fractionation in SP during N
2
O production has been reported.
56
  
Furthermore, while fractionation 
during N
2

15

18
O can be substantial, the effect of reduction on SP is 
generally quite small.
59
  
On t
his basis, the difference in SP observed in pure culture
60
 
and 
purified enzyme experiments
61
 
between bacterial denitrification (including nitrifier
-
denitrification, i.e. the reduction of nitrite to N
2
O under O
2
-
limiting conditions by nitrifiers) of 
-
10

has been used to estimate the relative production of N
2
O from these two pathways.
60a
  
Likewise, 
the SP for N
2

55c
  
Nonetheless, considerable variation in the SP values for specific processes exists, and this 
variation is best explained by differences in isotope fractionation associated with specific step
s in 
the nitrification and denitrification pathways.  In particular, the NO reduction step is especially 
93
 
important in controlling SP because this is the step where two NO atoms are assembled to create 
the N
2
O molecule.
62
 
Bacteria and fungi are the two major producers of N
2
O in the soil.  N
2
O produced by 
bacteria, however, is not necessarily an end product because it can be subsequently consumed by 
the bacterial nitr
ous oxide reductase (i.e. N
2
O can either be an end product or an intermediate in 
bacterial systems).  Conversely, most fungal organisms do not contain nitrous oxide reductase 
and therefore produce N
2
O only as an end product.
63
  
Based on the low SP values observed in 
N
2
O evolving from soils, bacterial denitrification is proposed to dominate N
2
O production in 
many environments.
58b
, 
64
  
However, there is a growing recognition of the importance of fungal 
denitrification in producing N
2
O
 
in some ecosystems, notably plantation forests and some 
grassland soils.
65
 
In this manuscript, we report the isotopic fractionation that occurs during the redu
ction of 
NO to N
2
O by purified fungal P450 nitric oxide reductase (P450nor) from 
Histoplasma 
capsulatum
.  By working with P450nor directly, we avoid the issue of compounding 
fractionation factors associated with multiple steps that is characteristic of mic
robial cultures, 
and we report the fractionation factors specific to NO reduction.  Our findings provide insight 
into the application of stable isotopes to reveal microbial production pathways as well as the 
reaction mechanism by which N
2
O is assembled by 
the P450nor enzyme.  Importantly, the amino 
acid sequence of 
H. capsulatum
 
P450nor is highly similar (approximately 60% identity) to other 
sequ
enced fungal P450nors (Figure 
16
), and it is therefore likely that our results will be generally 
applicable to th
is entire class of enzyme.  
 
94
 
 
Figure 
16
:
  
Multiple sequence alignment of P450nor(s)
 
from 
Trichosporon cutaneum
 
(
T. cutaneum
), 
Trichosporon asahii
 
var. asahii CBS 8904 (
T. asahii
8904), 
Trichosporon asahii
 
var. asahii CBS 2479 (
T. 
asahii
2479), 
Fusarium lich
enicola
 
(
F. lichenicola
1 and 
F. lichenicola
2), 
Fusarium oxysporum
 
(
F. 
oxysporum
), 
Histoplasma capsulatum
 
(
H. capsulatum
), 
Aspergillus oryzae
 
(
A. oryzae
).  All sequences 
were obtained from NCBI (
www.ncbi.nlm.nih.g
ov/
) except for 
H. capsulatum
 
P450nor (47
-
450) which 
 
95
 
Figure 16 (cont

d)
 
was obtained from Chao et al. (
Arch. Biochem. Biophys. 
2008,
 
480
, 132
-
137)
.  The multiple sequence 
alignment was performed using Clustal Omega (
www.ebi.ac.uk/Tools/msa/clustalo/
) and ESPript 3 
(
espript.ibcp.fr/ESPript/ESPript/
)
.
 
 
Methods
 
Expression and purification of P450nor.  
The P450nor gene (
NOR1
) from 
Histoplasma 
capsu
latum
 
was expressed as a C
-
terminal 6x
-
His
-
tagged protein on a pCW/Nor1p vector kindly 
provided by Prof. M. A. Marletta (Scripps Research Institute).
66
  
The plasmid 
was transformed 
into 
Escherichia coli
 
JM109
 
and a single colony was used to inoculate a 100 mL starter culture 

following morning, a 2 L flask with 1 L of Luria
-
Bertani (LB) medium was inoculated with the 
ove
rnight culture to an OD
600nm
 
of 0.1 and shaken at 37 °C.  After an OD
600nm
 
of 0.6 was reached, 
isopropyl
-
-
D
-
1
-
thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and 
the culture was shaken overnight at 25 °C.  P450nor was isolated as d
escribed by Chao 
et al
.
66
 
and purified using 1 mL of Ni
-
NTA Agarose resin (QIAGEN, Valencia, CA) in a 5 mL 
polypropylene column.
 
P450nor activity assay.  
H. capsulat
um
 
P450nor reduces NO to N
2
O according to the 
following reaction:
66
 
2NO + NADH + H
+
 

2
O + NAD
+
 
+ H
2
O
 
96
 
All activity assays were performed under anaerobic conditions.  Triplicate samples were mixed 
in 250 mL sterilized serum vials (Wheaton Glass, Milville, NJ) sealed with butyl rubber septa 
(Geo
-
Microbial Technologies, Ochelata, OK).  NO 
gas (99.5% purity, Airgas) was purified 
overnight by exposure to preconditioned (heated under vacuum at 300 °C) molecular sieves (5 
Å
) 
to reduce the N
x
O
y
 
background to less than 1%.  Purified NO (1 mL) was injected using a gas
-
tight syringe (Hamilton) into
 
a 250 mL glass seru
m vial (Wheaton Glass) containing 20 mL of 

-
nicotinamide adenine 

P450nor, and the progress was monitor
ed by removing 3 mL of headspace gas and injecting it 
onto a Shimadzu Greenhouse Gas Analyzer gas chromatograph (model GC
-
2014, Shimadzu 
Scientific Instruments, Columbia, MD) equipped with an electron capture detector (ECD) and a 
Hayesep N separation colum
n (GC oven temperature = 100 °C, ECD detector temperature = 
350 °C, N
2
 
carrier gas at a flow rate of 25 mL/min and 5% CH
4
/95% Ar make
-
up gas (Airgas) at 
a flow rate of 2.5 mL/min).  Samples were incubated on their side with shaking at room 
temperature for 
60 min, and a headspace sample was taken every 5 min.  To maintain the 
headspace at atmospheric pressure in the reaction vials, 3 mL of ultra
-
high
-
purity (UHP) N
2
 
was 
injected into the vial prior to removal of each 3 mL sample using a 5 mL gas tight glass 
syringe 

software.  A commercial 1 ppm standard (Scott Specialty Gases, Plumsteadville, PA), laboratory 
air, and standards prepared from UHP N
2
O and UHP N
2
 
tanks (Airgas) w
ere used for the 
calibration and calculation of the headspace concentrations of N
2
O.  
 
IRMS measurements.  
Isotope ratio data for N
2
O was obtained using a Trace Gas 
sample introduction system (Elementar Americas, Inc., Mount Laurel, NJ) interfaced to an 
97
 
Is
oprime isotope ratio mass spectrometer (IRMS) (Elementar Americas, Inc.).  The ion source 
settings in the mass spectrometer were optimized to provide precisions better than 0.5, 0.5, 0.7, 
0.7, and 1.2


15

18

15
N


15
N

 
and SP, respectively, for sample quantities between 6 
and 20 nmol.  The Trace Gas system uses He as the carrier gas, and CO
2
 
and H
2
O are removed 
from the sample gas by passage through Carbosorb and magnesium perchlorate traps (
Costech 
Analytical Technologies Inc, Valencia, CA), respectively.  The N
2
O in the sample gas was 
purified and concentrated via cryo
-
focusing followed by chromagraphic separation on a Poroplot 
Q column (Agilent, Foster City, CA).  Based on controlled fragme
ntation studies, the relative 
abundance of the 
14
N
15
N
16

15
N
14
N
16

2
O
+
 
and NO
+
 
ions in a single run as described by Sutka 
et al.
 

15
N

-
N
2
O and 

15
N

-
N
2
O were obtained following mass overlap correction as presented in Toyoda and 
Yoshida.
67
  
The concentration of N
2
O was dete
rmined using the area under the mass 44 N
2
O 
peak.  

15

15
N


18
O values of our laboratory pure N
2


 
 
Isotope value notations.  
The relative abundance of 
15
N and 
18
O in N
2
O molecules is 

 

sample
/R
standard
) 

 
1] × 
1000 
 
where:
 
R
sample
 
= 
15
N/
14
N or 
18
O/
16
O for the N
2
O in the samples
 
R
standard
 
= 
15
N/
14
N or 
18
O/
16
O for the N
2
O in the standards.   
 
The N and O standards are atmospheric N
2
 
and VSMOW,
37
 
respectively.
 
98
 
Site preference is defined as the difference between the isotope values of N

 
a
nd N

 
in N
2
O, as 
described in the following equation:
 

15
N

 
-
 

15
N

 
where:
 






















 






















 
and where 
15
R
 
and 
15
R
 
are defi
ned as:
 
15
R
 
= [
14
N
15
N
16
O/
14
N
14
N
16
O]
 
15
R
 
= [
15
N
14
N
16
O/
14
N
14
N
16
O]
 
All results are expressed as the mean ± standard deviation (SD).  
 
Calculations for determining enrichment factors (

) and kinetic isotope effects (KIE)
 
Microbial denitrification proceeds by
 
the progressive reduction of NO
3
-
 
to NO
2
-
 
to NO to 
N
2
O and finally to N
2
. 
 
Each step in this sequence, including diffusion of substrates into and out 
of cells, can be expected to 
discriminate against one isotope over the other depending on the 
specific re
action sequence.  This, in turn, will give rise to different isotope fractionation patterns 
depending on the types of organism and the specific reaction pathway
.  Here w
e follow the 
convention of Mariotti 
et al.
68
 
by def
ining the magnitude of isotopic fractionation during a single 

 

 
= k
2
/k
1
 
 
 
 
 
 
 
 
 
 
(1)
 
99
 
where k
1
 
and k
2
 
are the reaction rates for the light and heavy isotopically substituted compounds, 
respectively (a
lthough some authors use the inverse of this ratio).  As defined by Mariotti 
et al.
,
68
 

38
 

1
/k
2
)
-
1
 
= (KIE)
-
1
 
 
 
 
 
 
 
 
 
(2)
 
We further define an isotopi

 

 
= (

 
-
 
1)*1000
 
 
 
 
 
 
 
 
 
(
3
)
 
that can be quantified by the following expression of the Rayleigh isotope fractionation model:
 

15
N
p
 

15
N
so
 

 

p/s
[(
f
ln
f
)/(1
-
f
)]
 
 
 
 
 
 
 
(4)
 
where 
f
 
is defined as the fraction of NO remaining (determine
d as the ratio of the calculated NO 
concentration using measured N
2
O concentration at any point in time divided by the initial NO 

p/s
 
is the isotopic enrichment factor for any substrate (s) converted to a product 

15
N
so
 
refers to 
the isotopic composition of the substrate prior to initiation of the 
reaction.
69
  
This expression can be plotted such that a linear regression fit to the data yields the 
enrichment factor (

) as the slope from which the fractionation factor
 
(

) can be calculated 
(Figures 
17
-
2
0
).  The kinetic isotope effect values (KIEs) were then calculated using the 
fractionation factor values according to Equation (2).  Due to the complexity of the x
-
axis (
-
[(
f
ln
f
)/(1
-
f
)]), however, Rayleigh fractionation 
plots are not intuitive with respect to the time 
course of the reaction.  Therefore, in this manuscript we present simplified graphs that depict the 
actual change of the isotope values vs. the percentage of substrate converted (1
-
f
).  While these 
graphs we
re not used to calculate either enrichment factors or kinetic isotope effects, they offer a 
100
 
better perspective of the dynamics of the different isotopologues as a function of the reaction 
time course.  
 
 
 
Figure 
17
:
  
Best fit line of the Rayleigh equation
 

15
N in N
2
O produced by P450nor
.  

biological replicates.  
 
 
101
 
 
Figure 
18
:
  

18
O in N
2
O produced by P450nor.
  
Th
e 

biological replicates.  
 
102
 
 
Figure 
19
:
  

of the three biological replicates.
  
(A) B

15
N

 
in N
2
O 

15
N

 
in N
2
O 
produced by purified P450nor.  
 
103
 
 
Figure 
20
:
  
Best fit line of the Rayleigh equation to site preference (SP) val
ues.
  
The 
enrichment factors were calculated using the slopes of the lines as outlined in Equation 4 for 
each of the three biological replicates.  
 
 
Results
 

15

18
O analyses for N
2
O produced by 
H. capsulatum
 
P450nor 
 
Prior to analysis of the isotopic
 
fractionation by P450nor from 
H. capsulatum
, activity 
assays were performed to ensure that the different P450nor preparations yielded consistent 
enzyme activity.  The reduction of NO to N
2
O was monitored by analyzing the headspace gas via 
GC
-
ECD in three 
separate enzyme pre
parations.  As shown in Figure 
2
1
, all three samples 
showed continuous N
2
O production and identical production profiles over approximately 60 min, 
highlighting the robustness and reproducibility of the reaction conditions.  Thus, these s
ame 
104
 
reaction conditions were employed during the analysis of N
2
O via isotope ratio mass 
spectrometry.  
 
 
 
Figure 
2
1
:
  
N
2
O production by 
H. capsulatum
 
P450nor over the course of 60 min.  
 
 
The isotope ratio values were measured for different isotopologues 
of N
2

15

15
N

, 

15
N

 

18
O).  As shown in Figure 
22
A, when half of the NO was consumed there was a 

15
N in the accumulated N
2
O relative to the initial N
2
O measured.  This is in 
contrast to what is expected in a normal isotope effect
 
in which the molecules containing the 
light isotopes are preferentially converted first, resulting in enrichment in the heavy isotope of 
the residual substrate.  As the relative abundance of heavy isotopes in the substrate pool slowly 
increases over time,
 
there is a concomitant progressive enrichment in the heavy isotopes in the 
produc

15
N in the accumulated N
2
O over time indicates that 
15
NO 
105
 
molecules were converted to N
2
O preferentially over 
14
NO molecules by P450nor.  Consistent 

15
N) and
 
kinetic isotope 
effect (
15
N
-
KIE) values (Table 
8
) for N
2
O production revealed an inverse isotope effect, with an 

15

15
N
-
KIE of 0.9862 (± 0.0016).  Conversely, the N
2
O became 
enriched in 
18
O during the course of the reaction (Figu
re 
22
B).  This gives rise to a normal 
isotope effect and an 
18

18
O) of 
-

effect (
18
O
-
KIE) of 1.0264 (± 0.0041).
 
 
106
 
 
Figure 
2
2
: 
 

15

18
O of N
2
O produced by P450nor as a function of the fraction o
f NO 
reduced (1
-
f
)

15
N of N
2
O plotted against the fraction of NO reduced for three samples.  (B) 

18
O of N
2
O plotted against the fraction of NO reduced for three samples.  
 
 
 
107
 
Table 
8
:
  

dividual biological 
replicates of 
H. capsulatum
 
P450nor.
  

linear regression.  The KIE values were calculated based on Equations 2 and 3 using the 
corresponding 

 
values.  Mass averaged values and standard
 
deviations are shown for all values.  
 
 
Sample #1
 
Sample #2
 
Sample #3
 
Average 
±
 
SD
 

15

 
13.6
 
15.8
 
12.6
 
14.0
 
±
 
1.6
 
15
N
-
KIE
 
0.9866
 
0.9844
 
0.9876
 
0.9862
 
±
 
0.0016
 

15
N


 
-
13.2
 
-
9.3
 
-
15.2
 
-
12.6
 
±
 
3.0
 
15
N

-
KIE
 
1.0134
 
1.0094
 
1.0154
 
1.0127
 
±
 
0.0030
 

1
5
N

(

 
35.2
 
37.4
 
35.7
 
36.1
 
±
 
1.2
 
15
N

-
KIE
 
0.9660
 
0.9639
 
0.9655
 
0.9651
 
±
 
0.0011
 

18

 
-
28.9
 
-
27.0
 
-
21.3
 
-
25.7
 
±
 
4.0
 
18
O
-
KIE
 
1.0298
 
1.0277
 
1.0218
 
1.0264
 
±
 
0.0041
 

 
-
43.1
 
-
42.3
 
-
45.4
 
-
43.6±1.6
 
SP
-
KIE
 
1.0450
 
1.0442
 
1.0476
 
1.0456±0.0018
 
 
Two impo

calculated represented the enzymatic conversion of NO to N
2
O by P450nor.  In the first 
experiment, a mixture of N
2
O, NAD
+
, and P450nor were incubated under conditions that 
simulat
ed the end point of our reactions to evaluate if the back reaction was occurring.  After 70 
minutes, there was no detectable enzyme
-
mediated consumption of N
2
O (Figure 
23
).  In the 
second experiment, equal concentrations of NO and N
2
O were incubated either
 
in the presence of 
NADH/NAD
+
 
or in the presence of enzyme without NADH/NAD
+
.  In both cases, any change in 
108
 
the isotope ratio of the N
2
O over time was not greater than the analytical precision of the 
instrument, demonstrating that all of the enzymatic comp
onents need to be present for the 
interconversion between NO and N
2
O (Figure 
2
4
).  Together, these control experiments establish 
that our observed isotope effects cannot be explained by either N
2
O consumption or non
-
enzymatic exchange reactions, and that o
ur calculated fractionation factors must therefore 
represent inherent properties of NO reduction by P450nor. 
 
 
 
Figure 
23
: 
 
Quantification of N
2
O in the headspace in both the presence and absence of 
P450nor.
  
The conditions were chosen to test if the back
 
reaction (i.e. N
2
O oxidation) was 
occurring, and the conditions therefore mimicked those anticipated at the end of the N
2
O 
production assay except that no NO was present.  The back reaction contained 6.1 mM NADH, 
0.7 mM NAD
+
, and 0.02 mg/mL 
H. capsulatum
 



 
109
 
 
Figure 
2
4
:
  
Characterizati
on of the N
2
O isotope values to test for the presence of NO
-
N
2
O 
exchange.
  
In sample #1 (

) and #2 (


N
2
O and NO were incubated with 1.35 mM NAD
+
 
and 1.35 mM NADH in the absence of 
P450nor for up to 90 mi
n.  In sample #3 (

), the same amounts of N
2
O and NO were incubated 
with purified P450nor (0.02 mg/mL), but neither NAD
+
 
nor NADH was added.  (A) 

15
N value 
of N
2
O measured during the course of 90 minutes. (B) 

18
O value of N
2
O measured for 90 
minutes. (C)
 
and (D) 

15
N

 
and 

15
N

 
values of N
2
O measured for 90 minutes (E) Site preference 
values of SP during the course of 90 minutes.  
 
110
 
Site preference values for N
2
O produced by 
H. capsulatum
 
P450nor 
 
To obtain a more detailed understanding of the isotopic pre
ference during NO reduction 
to N
2
O by P450nor, we calculated the fractionation for both N

 
and N

.  In general, the isotope 
ratio of the N

 
in N
2
O exhibited a relatively modest change as the reaction progressed.  While we 
have no clear explanation for the 



 
from 
-

-

between 15
-
55% NO reduction, indicating that there is a small preference for the light isotope of 
nitrogen to be inc

effect for N


-


15
N

 

ive to the N
2
O 
initially produced (Figure 
2
5
B), indicating that 
15

position of N
2
O.  Therefore, N

 


ue of 0.9651 (± 0.0011) (Tab
le 
8
).  Overall, these results indicate that during 
the course of the reaction, 
15
N was strongly preferred over 
14
N in the N

 
position when N
2
O was 
formed, while a weaker reverse pattern was observed in the N

position.  The bulk N 
fractionation factor is 

-
 

-

15
N values and therefore the isotope effect for 

 
111
 
 
Figure 
2
5
:
  

15
N

 

15
N

 
of N
2
O produced by as a function of the fraction of NO reduced 
(1
-
f
).
  

15
N

 
of N
2

15
N

 
of N
2
O plotted against the fraction of NO reduced for three samples.  
 
 
The SP of N
2

b
y the time half of the NO was reduced, giving rise to an enrichment in 
15

2
6
).  
112
 

15
N

 

15
N

 
during the 

c isotope effect (SP
-
KIE) for the 
SP were calculated to be 
-

during N
2
O production SP has a normal isotope effect.  In contrast, previous results using pure 
cultures revealed no change in S
P over time,
55c
 
which highlights the importance
 
of using purified 
enzymes to measure individual steps in the denitrification pathway.
 
 
 
Figure 
2
6
:
  
Site preference (SP) values of N
2
O versus the fraction of NO reduced (1
-
f
).
  
Three independent biological replicates are shown.
 
 
Discussion
 
Fractionation 
during reduction of NO by 
P
450nor
 
113
 
The typical behavior of a kinetic isotope effect in an enzymatic reaction is for the light 
isotope to be preferentially transferred to the initial product followed by progressive enrichment 
in the abundance of the heavy is
otope as a function of time or with the extent of the reaction.  

15
N


18
O 
and SP.  By the Rayleigh fractionation model described in Equation 4, normal fractionation 
resul
ts in negative isotopic e
nrichment factors (Table 
8
).  Inverse isotope effects, in which the 
product becomes preferentially depleted in the heavy isotope as a function of the extent of the 
reaction, are rarely observed in enzymatic reactions,
70
 

15
N 

15
N

 
during NO reduction (Table 
8
).  Similar to our findings is the observation of an inverse 
isotope effect associated with formation of the N
-
O bond during microbial nitrification,
70
 
although in our study the inverse isotope effect is associated with the binding of the first NO 
molecule (whose N atom ultimately becomes N

 
in N
2
O)
71
 
to the Fe active site.  An inverse 
isotope effect is
 

15

15

15
N of the 
N

 
and N

 

15
N is caused by the greater isotopic 
enrichment factor for the N

 
position that is greater and opposite in direction t
o that associated 
with the N

 
position.  
 
Two possible trivial explanations could account for an apparent inverse isotope effect 
during the reduction of NO to N
2
O.
70a
  
The first, enzyme
-
level reversibility, results if the reverse 
reaction fractionates to a greater extent than the forward reaction and can only be significant if 
there is sufficient mass flux in t
he reverse direction to express the fractionation.  We exposed 
N
2
O to the identical conditions we used to catalyze NO reduction and observed no enzyme
-
catalyzed decrease in the abundance of N
2
O with time (Figure 
23
). Consequently, we observed 
no indication
 
of the reverse reaction under our experimental conditions.  Secondly, isotopic 
114
 
equilibrium between the substrate of the reaction and another species in the system prior to 
enzymatic reduction could also result in the appearance of inverse fractionation.  
For example, a 
pH dependent pre
-
equilibrium between H
2
S and HS
-
 
was used to explain an inverse fractionation 
during anaerobic sulfide oxidation.
72
  
The only other N species of relevance in our exper
iments is 
N
2
O and we allowed NO and N
2
O to equilibrate for 90 minutes and observed no changes in the 
isotopic composition of N
2
O (Figure 
2
4
).  Consequently, we conclude that isotopic equilibration 
is not a factor driving our experimental results.  
 
Based o

nitrogen atom must result from an inherent property of the enzyme reaction mechanism.  Simply 
stated, the N atom must be more strongly bonded in the transition state than in th
e ground state 
of the reaction.  This has been proposed to be the case for the inverse isotope effect observed in 

15

18
O during the oxidation of nitrite to nitrate during microbial nitrification.
70
  
Conversely, if the N atom is more strongly bonded in the substrate than in the transition state, 
then a normal isotope effect is observed.  This is the case for most N oxide reducing reactions 
(nitrate red
uction, nitrite reduction and nitrous oxide reduction) during the denitrification 
process.
53b
, 
54a
, 
59
  
Our case is distinct, however, in that we observe an inverse isotope effect only 
for the 

 

 
Implications for the reaction p
athways of NO reduction by P450nor
 
Two scenarios have been proposed to explain the SP values in N
2
O resulting from 
reduction of NO that involve either simultaneous or sequential binding of the two N atoms to the 
Fe centers of bacterial or fungal NOR.
58a
, 
62a
  
If the two N atoms bind simultaneously, then little 
isotopic preference is expected between the N

 
and N

 

115
 
values for N
2
O production via bacterial denitrification range between approximately 
-


58
, 
64
 
and are consistent with simultaneous binding.  Although the precise catalytic mechanism 
of bacterial nitric oxide reductase remains uncertain, a reasonable hypothesis is that one NO 
binds to the non
-
heme iron while the
 
other NO binds to the heme iron.
73
  
P450nor, however, 
contains only a single heme iron at the active site,
74
 
and studies have clearly indicated that each 
NO molecule binds separately to the enzyme prior to formation of the N=N bond.
75
  
Isotopic 
discrimination between the N atoms could occur in sequential binding if the first molecule of NO 
(whose nitrogen atom becomes N

 
in N
2
O)
71
 
binds with a strong preference for 
14
N followed by 
the slower binding of the secon
d NO molecule that experiences a smaller degree of isotopic 
segregation.
62a
  
However, this does not match the inverse kinetic isotope effect we observed for 
N

 
(KIE = 0.9651).  In addition, this would result in a positive SP value that trends toward zero 
over time, and our results indicate that SP becomes mo
re positive over time.  Therefore, we 

heme center of P450nor results in a more tightly bonded N atom in the transition state than the 
ground state, which lead
s to an inverse 
kinetic
 
isotope effect (Figure 
27
).  In addition, the N atom 
of this enzyme
-
NO complex has a higher bond order than the free NO molecule itself,
76
 
and it is 
known that this reaction step is freely reversible,
77
 
both of which give rise to an inverse 
equilibrium
 
isotope effect.  Thus, we propose that it is this combination of kinetic and 
equilibrium inverse isotope effects 
that gives rise to the trend of SP becoming more positive over 
time.
 
116
 
 
Figure 
27
:
  
An energy diagram depicting the proposed binding of the first NO to the 
P450nor heme active site.
  
The bond order increases from the ground state to the transition 
state
82
 
leading to a steeper energy well and a larger isotopic difference in zero
-
point energy (ZPE) 
in the transition state.
83
  
The magnitude and type (i.e. normal or inverse) of the isotope effect is 

te and the transition state.
 
 
The addition of the second NO molecule, whose N atom ultimately becomes N
, 
involves the conversion of an Fe(IV)
-
NHOH
-
 
complex to a protonated Fe(III)
-
N
2
O
2
H
2
 
species.
71
  
It is likely that this is the rate
-
limiting step between the binding of the
 
second NO molecule and 
release of N
2
O.  In contrast to the observations of a

18
O during oxidation of nitrite,
70b
 
we 
observe a normal isotope effect during production of N
2
O via P450nor.  This observation is 
consistent with t
he oxygen atom in N
2
O being derived from the second NO molecule, whose 
nitrogen atom ultimately becomes N

.  
 
 
117
 
Implications for isotope source tracing of N
2
O production
 

15

15
N

 
as well as normal 

18
O are in contrast with the isotope effects observed during N
2
O 
production via nitrite r
eduction in two species of fungi that utilize the P450nor enzyme.
55c
  
No 

18
O and SP during fungal denitrification using pure 
culture whereas substantial isotope effects were found in this study during NO reduction by 
purified P450nor.  While an isotope effect dur
ing fungal denitrification in the N

 
and N

 
positions was not reported by Sutka 
et al.
,
55c
 
two of the four cultures showed evidence of 

15
N

 
of 
-
62.6 and 
-

15
N

 
of 
-

15
N

 
in Sutka 
et al.
55c
 
are limited, there 
is evidence of an inverse isotope effect of comparable magnit
ude to what we observe with the 
P450nor enzyme from 
H. capsulatum
.  Sutka 
et al.
55c
 
also observed markedly large variation in 

15
N of 
-
74.7 to 
-

cell to limit expression of the enzymatic fractionation during NO reduction i
n fungi.
78
  
Indeed, a 

15
N and the rate constant was observed which is consistent with diffusion 
control of both the reaction rate and the net isotopic fractionation.  Consequently, the contrast in 
the direction and magnitude of fractionation fac
tors for N
2
O production between the entire 
fungal denitrification process and those of purified fungal P450nor may be the result of variable 
expression of the small fractionation imposed by diffusion of substrates into and out of cells.  In 
other words, th
e large isotope effect associated with enzymatic reduction can only be expressed 
when diffusion does not limit the supply of NO to the enzyme.  When diffusion is limiting, 
isotopic discrimination by the enzyme cannot be expressed as all available NO to the
 
enzyme is 
reduced.
56
  
An additional isotope effect is undoubtedly impose
d by the reduction of nitrite to NO 
118
 
during fungal denitrification that was also not a factor in this current study because we used NO 
as the substrate.  Because there is a tendency for the most rate limiting step in denitrification to 
control the expressed
 
fractionation, the fractionation observed in fungal denitrification may 
largely have been controlled by diffusion, nitrite reduction to NO, and/or reduction of NO.  
Further, it is conceivable that the rate
-
limiting step changed during the course of the fu
ngal 
culture reactions or that the fractionation of the individual steps (NO
3
-
 

2
-
 

2
O) 
partially cancel one another.  Thus, compared to experiments utilizing microbial cultures, it is 
likely that our results more accurately describe the isotope 
effects associated specifically with 
NO reduction to N
2
O catalyzed by the P450 enzyme. 
 
 
Nonetheless, it is recognized that SP must be controlled by the NO reduction step given 
that it is during this process that two NO atoms are joined to produce the line
ar, asymmetric N
2
O 
molecule.
62
  
Schmidt 
et al.
 
(2004)
62b
 
proposed that SP would invariably experience fractionation 
given the unique pathway of formation for each N atom in N
2
O by both P450nor and bacterial 
nitric oxide reductase, but no supporting data was provided.  Our study clearly demonstrates 
that 
there is substantial fractionation in SP during N
2
O formation from the P450nor enzyme.  In other 

15
N

 

15
N

 
are not equivalent, which is in 
contrast to what has been observed in microbial culture
.
55c
, 
58
  
Curiously, no alteration was 
observed in SP during 
in vivo
 
N
2
O production in pure culture, while we o
bserved a clear trend 
in 
vitro
 
with purified P450nor.  Because the two N atoms in N
2
O combine during the NO reduction 
step, SP values should be independent of the steps preceding NO reduction, and one would 
therefore expect 
in vivo
 
and 
in vitro
 
studies to 
give similar results.  Although the reduction of 
N
2
O to N
2
 
is known to affect SP,
59
 
the species of fungi cultured in Sutka 
et al.
55c
 
lack the ability 
to reduce N
2
O, and this can therefore not explain the diffe
rent SP results. 
 
119
 
Interestingly, the SP values obtained near the end of the P450nor experiment (with an 

26
) which is 

et al.
55c
  
Extrapolation of our data to 100% 
conversion indicate
s that SP values during NO reduction by the fungal NOR enzyme could reach 
55
-

reduction whereas in pure fungal cultures the SP values during nitrite reduction were nearly 

by 
linear regression in Figure 
2
5) we obtain an extent of conversion (1
-
f
) within the fungal cultures 
of approximately 65%.  This seems to imply that the rate of n
itrite and NO reduction are the 
same (thereby maintaining a steady NO concentration in the cell), and that the magnitude of the 
P450nor NO binding constant (
K
d
) maintains (1
-
f
) at 65%.  Nonetheless, the observation of an 
inverse isotope effect in one cultu
re of fungi in Sutka 
et al.
55c
 
indicates that n
itrite and NO 
reduction do not work in harmony under all circumstances.  
 
 
The observation of constant SP values in microbial cultures even as substrates are 
depleted
58b
, 
79
 
has led some to suggest that SP could be used as a conservative tracer of N
2
O 
production, and in fact, this approach has been used in a number of studies.
60a
, 
80
  
Indeed, the 
predominance of low SP values for N
2
O evolving from soils provides a strong indication that 
bacterial denitrification is the 
predominant microbial source of this gas in many terrestrial 
ecosystems.
48b
, 
60
  
Our results, however, clearly 
indicate that fractionation of SP during N
2
O 
production by P450nor is not zero, and that SP values higher and lower than the proposed end 

should be taken when one 
tries to determine the sources of N
2
O production under different field 
settings as various growth conditions (i.e. pH, moisture level, nutrient source, etc.) might perturb 
120
 
the flux in the denitrification pathway, thereby altering the intracellular NO conce
ntration and 
resulting in a change of the SP value.  Nonetheless, the observation of constant SP in pure 
culture implies a steady NO concentration is maintained in the cell under many growth 
conditions.
 
In summary, isotopic fractionation patterns during NO
 

2
O) catalyzed 
by fungal P450nor were analyzed in detail.  The O and N

 
showed normal isotope effects while 

15

15
N

 
exhibit inverse isotope effects.  These data are consistent with the 
sequential binding and reduction react
ion mechanism proposed for P450nor.  The high sequence 
identity observed among fungal P450nors suggests that these isotopic fractionation patterns may 
be characteristic of this entire class of enzyme.  Interestingly, the SP values showed isotopic 
enrichmen
t during the course of the reaction, indicating that the SP value associated with a 
specific pathway may not remain constant in pure microbial culture or in the field.  
 
Acknowledgement
 
The research was funded by the National Science Foundation (EAR
-
105343
2).  We 
would like to thank Kateri Salk (Michigan State University) for her help in setting up the GC
-
IRMS for the analysis of NO
-
N
2
O exchange reaction and Nicolai Lehnert (University of 
Michigan) for helpful discussions.  
 
 
 
121
 
 
 
 
 
 
 
 
 
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CHAPTER 5
 
CONCLUSIONS AND FUTURE DIRECTIONS
 
 
130
 
In this thesis I demonstrated that by using stable isotopes and purified hydrogenase enzymes, the 
isotope fractionation factors can be measured and d
etermined with the help of isotope ratio mass 
spectrometry (Chapter 2).  Hydrogenases are enzymes that catalyze the reversible formation of 
H
2
,
 
which can be used as a clean and renewable energy.  H isotopes have the potential to be a 
powerful tool in quant
ifying proton flux in hydrogenase
-
catalyzed reactions because the 
movement of H ions is involved in almost every essential step of the mechanism.  However, 
because H
2
 
is highly diffusive, stable H isotopes have thus far not been widely used in studying 
bio
logical H
2
 
production.  Our proof
-
of
-
concept experiment provided the first efficient method to 
quantify the fractionation factors of the hydrogenase
-
catalyzed H
2
 
production process.  
 
After establishing the method to measure the fractionation of hydrogenas
e
-
catalyzed reactions, I 
utilized this approach to determine the fractionation pattern for a number of [NiFe]
-
 
and [FeFe]
-
hydrogenases (Chapter 3).  I ascertained the isotope fractionation patterns for each of the three 
reactions catalyzed by hydrogenases:
 
H
2
 
production, H
2
 
consumption, and H
2
-
H
2
O exchange.  
The measurements and analyses were used to facilitate our understanding of hydrogenase 
catalysis under physiological conditions where three reactions can occur simultaneously.  In 
addition, the fraction
ation factors determined for a specific reaction (i.e. production, 
consumption, or H
2
-
H
2
O exchange) for different hydrogenases can be used as signatures in 
studying H
2
 
metabolism in complex microbial communities where multiple hydrogenases coexist.  
In ess
ence, the amount of H
2
 
in a microbial community is the composite of quantifiable 
contributions from different hydrogenases.  Using a multiple component mixture equation (as 
exemplified in the work published by Kreuzer et al.
1
) and the fractionation factors for each 
hydrogenase, one can potentially quantify the 
fractions of H
2
 
produced or consumed by different 
hydrogenases in a microbial community.  
 
 
131
 
Our analysis of the hydrogen production results indicated that all six hydrogenases displayed 
normal isotope effects, which indicated that the H
2
 
produced was severe
ly depleted in 
2
H relative 
to the substrate water.  This result was consistent with the fact that deuterons formed stronger 
and less labile hydrogen bonds during H
2
 
production.  More importantly, the fractionation factors 
for each class of hydrogenases app
eared to cluster, which was consistent with the difference in 
active site structures between [FeFe]
-
 
and [NiFe]
-
hydrogenases.  In our analysis, the three 
[NiFe]
-

-
 
0.40) turned out to be statistically smaller than the three [FeFe]
-
hyd

-
 
0.55).  The results from the H
2
 
production study support our original 
hypothesis that fractionation factors can be used as signatures for different hydrogenases in the 
study of H
2
 
source partitioning in a complex microbial community w
here there are multiple 
hydrogenases.
 
In addition, the isotope fractionation patterns for H
2
 
consumption and H
2
-
H
2
O exchange were 
also determined.  In our analysis, the fractionation factors determined for H
2
 
consumption were 
approximately 0.88, consistent
 
with metal
-

2
-
H
2
) 
complexes which displayed normal equilibrium isotope effects.  The fact that the fraction factors 
were similar was surprising to us.  We predicted that because of the distinct active site structures
 
of [FeFe]
-
 
and [NiFe]
-
hydrogenases, that H
2
 
consumption would have different fractionation 
factors.  We hypothesize that 0.88 is a net fractionation factor, and that steps other than H
2
 
oxidation (e.g. H
2
 
diffusion, H
2
 
consumption step, proton transfer, e
lectron transfer, etc.) must be 
masking the true fraction factor.  Nevertheless, the fractionation factors of the H
2
 
consumption 
process indicated that hydrogenase
-
catalyzed H
2
 
consumption exhibited a normal isotope effect, 
with hydrogen reacting faster th
an deuterium.  Interestingly, we observed no obvious isotope 
 
132
 
fractionation in the H
2
-
H
2
O exchange experiments during the three
-
hour period of the reaction.  
Thus, under our reaction conditions, H
2
-
H
2
O exchange is not occurring to a significant extent. 
 
In 
addition to the study of purified hydrogenases, we also quantified the isotopic fractionation of 
purified fungal nitric oxide reductase, an enzyme that produces N
2
O via the reduction of NO.  
Fungal nitric oxide reductases are generally called P450nor becau
se of the involvement of a 
P450 heme in the active site.  In this study, we used gas chromatography
-
isotope ratio mass 
spectrometry (GC
-

15

18

15
N


15
N

 
of N
2
O generated by 
purified fungal P450nor from 
Histoplasma capsulatu
m
.  These isotope values can provide us the 
information needed for both the calculation of fractionation factors and site preference values as 
well as the clarification of the reaction mechanism.  First, the isotope values were used to 
calculate the fracti
onation factors, which indicated that O and N

 
displayed normal isotope 
effects during enzymatic NO reduction.  However, bulk N and N

 
showed inverse isotope effects.  
Importantly, the observed inverse isotope effect in N

 
provided support for reversible binding of 
the first NO in the proposed P450no
r reaction mechanism.  In addition, the isotope values were 

15
N

 

15
N

.  
The site preference value is a key index for identifying the sources and sinks of N
2
O.  In our 
exper
iments, however, we found that in contrast to the results reported from pure microbial 
culture studies, the site preference value was not constant in our purified enzyme analyses.  
These results suggested that site preference for microbial cultures can var
y depending on the 
growth conditions, which may complicate source tracing during microbial denitrification.  
 
Our study of the P450nor was well received by the scientific community, which prompted us to 
initiate a collaboration with a computational chemist
.  The information from the fractionation 
patterns determined in our P450nor studies was shared with Professor Nicolai Lehnert at The 
 
133
 
University of Michigan.  It is hoped that his expertise in computational modeling of the 
thermodynamics and reaction inter
mediates in N
2
O biosynthesis
2
 
can be combined with our 
observed fractionation factor values in P450nor catalyzed NO conversion
,
3
 
which will shed light 
on the details of the P450nor reaction mechanism.  
 
Very recently, experiments using a similar approach were designed and initiated to determine 
the 
fractionation patterns for bacterial nitric oxide reductase.  In addition to fungal P450nor, this 
enzyme is also a major player in microbial N
2
O generation.
4
  
The analysis of this enzyme will 
not only contribute to our understanding of N
2
O cycling
 
in the environment, but also to the 
mechanism of bacterial N
2
O biosynthesis.  
 
In summary, we have demonstrated that stable isotopes can be a powerful tool in studying 
enzyme catalysis.  In particular, the study of isotopic fractionation patterns of purif
ied enzyme
-
catalyzed reactions can provide important information about reaction mechanisms (Chapter 2, 3, 
and 4) as well as intracellular metabolic homeostasis (Chapter 4).  
 
 
 
 
134
 
 
 
 
 
 
 
 
 
BIBLIOGRAPHY
 
 
 
 
135
 
BIBLIOGRAPHY
 
 
 
1.
 
Kreuzer
-
Martin, H.
 
W.; Ehleringer, J. R.; Hegg, E. L., Oxygen isotopes indicate most 
intracellular water in log
-
phase 
Escherichia coli
 
is derived from metabolism. 
Proc. Natl. Acad. Sci. U.S.A. 
2005,
 
102
 
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-
17341.
 
2.
 
McQuarters, A. B.; Wirgau, N. E.; Lehnert, N., Mo
del complexes of key intermediates in fungal 
cytochrome P450 nitric oxide reductase (P450nor). 
Curr. Opin. Chem. Biol. 
2014,
 
19
 
(0), 82
-
89.
 
3.
 
Yang, H.; Gandhi, H.; Ostrom, N. E.; Hegg, E. L., Isotopic fractionation by a fungal P450 nitric 
oxide reductase 
during the production of N
2
O. 
Environ. Sci. Technol. 
2014,
 
48
 
(18), 10707
-
10715.
 
4.
 
Baggs, E. M., A review of stable isotope techniques for N
2
O source partitioning in soils: recent 
progress, remaining challenges and future considerations. 
Rapid Commun. Mas
s Spectrom. 
2008,
 
22
 
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1664
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1672.