From the Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322
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ABSTRACT |
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The nitrogenase catalytic cycle involves binding
of the iron (Fe) protein to the molybdenum-iron (MoFe) protein,
transfer of a single electron from the Fe protein to the MoFe protein
concomitant with the hydrolysis of at least two MgATP molecules,
followed by dissociation of the two proteins. Earlier studies found
that combining the Fe protein isolated from the bacterium
Clostridium pasteurianum with the MoFe protein isolated
from the bacterium Azotobacter vinelandii resulted in an
inactive, nondissociating Fe protein-MoFe protein complex. In the
present work, it is demonstrated that primary electron transfer occurs
within this nitrogenase tight complex in the absence of MgATP (apparent
first-order rate constant k = 0.007 s The metalloenzyme nitrogenase, which catalyzes the
biological reduction of nitrogen (N2) to ammonia
(NH3), is composed of two separable component proteins (1).
One component, the molybdenum-iron (MoFe)
protein,1 is an
MgATP appears to play several key roles in the reduction of
substrates by nitrogenase (8). First, the binding of MgATP to the Fe
protein induces conformational changes within the Fe protein (10).
These conformational changes influence the binding of the Fe protein to
the MoFe protein (11). Once the two proteins have associated, MgATP
hydrolysis functions somehow to facilitate electron transfer from the
Fe protein to the MoFe protein. Finally, the hydrolysis of MgATP to
MgADP and Pi is believed to be involved in stimulating the
dissociation of the Fe protein from the MoFe protein (12-14).
Earlier studies (15-17) found that combining the Fe protein isolated
from the bacterium Clostridium pasteurianum (Cp2) with the
MoFe protein isolated from the bacterium Azotobacter
vinelandii (Av1) resulted in a nondissociating complex (Cp2·Av1)
(see Equation 1).
1)
and that MgATP accelerates this electron transfer reaction by more than
10,000-fold to rates comparable to those observed within homologous
nitrogenase complexes (k = 100 s
1).
Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the
Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein
P2+/N cluster were determined for both the
uncomplexed and complexed proteins and with or without MgADP.
Calculations from electron transfer theory indicate that the measured
changes in Em are not likely to be sufficient
to account for the observed nucleotide-dependent rate
accelerations for electron transfer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2 tetramer containing two
[7Fe-Mo-9S-homocitrate] cofactors (FeMoco), which are the site of
substrate reduction, and two P or [8Fe-7S] clusters, which are
believed to mediate electron transfer to FeMoco (2, 3). The other
component, the iron (Fe) protein, is a homodimer containing a
single [4Fe-4S] cluster bridged between the subunits and two
nucleotide binding sites (4, 5). The current model for the catalytic
mechanism of nitrogenase holds that the reduced Fe protein, with two
bound MgATP molecules, binds to the MoFe protein, and a single electron
is transferred from the Fe protein to the MoFe protein in a reaction
that is somehow coupled to the hydrolysis of two MgATP molecules (6).
The oxidized Fe protein, with two molecules of MgADP bound, then
dissociates from the MoFe protein (7). Several cycles of component
protein binding, MgATP hydrolysis, and intercomponent electron transfer are required to reduce substrates by multiple electrons (8, 9).
(Eq. 1)
The association constant (Ka) for the Cp2·Av1 complex was determined to be approximately 10-fold higher than that for the homologous A. vinelandii nitrogenase complex and 100-fold higher than that for the homologous C. pasteurianum nitrogenase complex (15). In addition, this protein-protein complex could form with or without nucleotides (17). Although the Cp2·Av1 complex is inactive in all substrate reduction activities, it was later found (18) that this complex could still hydrolyze MgATP to MgADP + Pi at low rates (36-fold lower than the homologous protein complexes).
In the present work, we present evidence that primary
electron transfer, but not subsequent electron transfer
reactions, occurs within the Cp2·Av1 tight complex. Primary
electron transfer was found to occur without added nucleotides
at low rates, but the addition of MgATP accelerated electron
transfer by more than 10,000-fold to rates near those
observed in the homologous protein complexes. How these
results relate to our current understanding of the roles of
MgATP in the nitrogenase mechanism is discussed.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Nitrogenase
Proteins--
Nitrogenase Fe and MoFe proteins were expressed in
A. vinelandii cells and purified to apparent homogeneity
essentially as described previously (19, 20). C. pasteurianum strain W5 (a gift from J.-S. Chen, Virginia Tech) was
grown under N2-fixing conditions essentially as described
(21), and the nitrogenase Fe protein was purified (22). Protein
concentrations were determined by a modified biuret method (23), with
bovine serum albumin as the standard, or by the visible absorption
using absorption coefficients of 11.1 mM1·cm
1 at 400 nm for reduced
Av2 and Cp2 (24), and 62.3 mM
1·cm
1 at 400 nm for reduced
Av1 (24, 25). All of the nitrogenase proteins used in this study had
specific activities of at least 1,800 nmol of acetylene
reduced·min
1·mg of protein
1. Protein
manipulations were performed in the absence of O2 in sealed
serum vials under an argon atmosphere or in an argon-filled glovebox
(Vacuum Atmospheres, Hawthorne CA).
UV-visible Absorption Spectra of Av2 and Cp2-- Absorption spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer in 2.0-ml quartz cuvettes fitted with serum stoppers. Anaerobicity was obtained by purging the cuvettes with O2-free argon for 6 min. The buffer used for these experiments was 100 mM MOPS, pH 7.0, with 2 mM dithionite. A MgATP-regenerating system was included, as indicated, during nitrogenase catalysis to prevent the formation of MgADP, which is a known inhibitor of MgATP binding to the Fe protein (26).
Oxidation of Av1--
The Av1 P clusters were oxidized by 2 eq
of electrons by the addition of an excess of oxidized indigo
disulfonate (27). This was done by exchanging dithionite-reduced Av1
into anaerobic, dithionite-free 100 mM MOPS buffer, pH 7.0, with 250 mM NaCl, by passage through a Sephadex G-25 column
in an anaerobic glovebox. Av1 was then oxidized to the P2+
state by the addition of an excess of oxidized indigo disulfonate (28),
and the mixture was allowed to incubate for 15 min. Indigo disulfonate
was then separated from Av1 by passage through a Dowex-1 (Sigma) column
equilibrated with 50 mM MOPS buffer, pH 7.0. The oxidized
Av1 (P2+) was collected, and the concentration was
determined from the absorption spectrum and the known absorption
coefficient of 73 mM1·cm
1 at
400 nm (29). Reduced, dithionite-free Cp2 was prepared by passage of
the protein through a Sephadex G-25 column equilibrated with 50 mM MOPS buffer, pH 7.0.
EPR Spectroscopy-- EPR spectra were recorded on a Bruker ESP300E spectrometer equipped with a dual-mode cavity and an Oxford ESR 900 liquid helium cryostat. In all cases, 4-mm calibrated quartz EPR tubes (Wilmad, Buena, NJ) were used. All spectra were recorded at 12 K with 1,024 points/scan. The conversion time and time constant were 10.24 ms. All other parameters are noted in the figure legends.
Stopped-flow Spectrophotometry-- Electron transfer from Cp2 to Av1 was monitored by the increase in the visible absorbance of Cp2 upon oxidation of the [4Fe-4S] cluster from the reduced (+) to the oxidized (2+) state (30). This electron transfer reaction was monitored in real time by use of a Hi-Tech SF61 stopped-flow spectrophotometer equipped with a data acquisition and curve fitting system (Salisbury, Wilts, U. K.). The SHU-61 sample handling unit was kept inside an anaerobic glovebox (Coy Products, Grass Lake, MI) with a gas atmosphere of 95% N2 and 5% H2 and an oxygen concentration less than 1 ppm oxygen. Reactant solutions were thermostatted to within ± 0.1 °C by means of an FC-200 Techne flow cooler attached to a closed circulation Techne C-85D water circulator (Techne Ltd., Duxford, Cambridge, U. K.). Data were collected at 430 nm for the oxidation/reduction of Fe protein. Earlier work demonstrated no significant change in the absorption coefficient of the MoFe protein at 430 nm resulting from electron transfer from the Fe protein (30). All reactions were carried out in 100 mM HEPES buffer, pH 7.4, with 2 mM dithionite. In all cases, reactions were initiated by rapidly mixing reactants contained in the two drive syringes of the stopped-flow instrument. The instrument mixing time was determined to be approximately 4 ms. Reaction conditions are noted in the appropriate figure legends.
Apparent first-order rate constants for electron transfer (kobs) were determined from nonlinear, least squares fits of the absorbance versus time traces to the equation for a single exponential. In all cases, the absorbance versus time traces represented the average of three consecutive experiments.
Redox Titrations--
Potentiometric redox titrations were
performed essentially as described previously (31) in 50 mM
Tricine buffer, pH 8.0, with 150 mM NaCl and a series of
redox mediators. For titrations of the Cp2 [4Fe-4S]2+/+
couple, the mediators included a 50 µM concentration each
of flavin mononucleotide (Em = 172 mV and
238 mV), benzyl viologen (Em =
361 mV),
methyl viologen (Em =
440 mV), and
N,N'-propane-2,2'-dipyridinium (Em =
590 mV; a gift from Dr. Vernon Parker,
Utah State University). For titrations of the Av1
P2+/N couple, the mediators included a 100 µM concentration each of flavin mononucleotide, benzyl
viologen, and methyl viologen. The reduction potential of the mediator
and protein solution was adjusted by the addition of a 5 mM
dithionite (Na2S2O4) solution or a
25 mM oxidized indigo disulfonate solution. For titrations
below
500 mV, reduction was accomplished in a standard H-cell using a
Hewlett-Packard 6212C constant power supply with a gold wire working
electrode and a platinum mesh counter electrode (2 × 1 cm). In
all cases, the reference electrode was an Ag/AgCl microelectrode that
was calibrated against a standard calomel electrode. All potentials are
reported relative to the normal hydrogen electrode. At defined
potentials, 250-µl aliquots were removed and were frozen in
calibrated quartz EPR tubes (Wilmad, Buena, NJ). The relative concentrations of the reduced and oxidized states for each metal center
were determined by the peak-to-peak height of the appropriate EPR
signal. Plots of the fraction of maximum signal intensity versus potential were fit to the Nernst equation (Equation 2) using the nonlinear, least squares fitting program Igor Pro
(Wavemetrics, Lake Oswego, OR) where E is the measured
potential, Em is the midpoint potential,
R is the gas constant, T is the temperature, n is the number of electrons transferred, F is
Faraday's constant, [red] is the concentration of the reduced
species, and [ox] is the concentration of the oxidized species
(32).
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(Eq. 2) |
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RESULTS |
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Electron Transfer in the Absence of Nucleotides Monitored by
UV-visible Absorption--
Electron transfer from the Fe protein to
the MoFe protein can be monitored by the increase in the electronic
absorption spectrum in the 430 nm region as a result of the oxidation
of the Fe protein [4Fe-4S] cluster from the 1+ to the 2+ oxidation
state (30). When dithionite-reduced Cp2 ([4Fe-4S]+) was
combined with dithionite-reduced Av1 in the absence of any nucleotides,
an increase in the absorbance in the 430 nm region was observed over 10 min as shown in Fig. 1A. This
is consistent with the oxidation of the [4Fe-4S] cluster of Cp2 to
the 2+ state resulting from electron transfer to the MoFe protein in
the absence of nucleotides. In contrast, when A. vinelandii
Fe protein (Av2) was mixed with Av1 under identical conditions, no
changes were observed in the 430 nm region of the absorbance spectrum
even after a 10-min incubation (Fig. 1B, traces 1 and 2). Only upon the addition of MgATP to the
Av2·Av1-containing solution did the absorbance in the 430 nm region
increase (Fig. 1B, trace 5), consistent with the
dependence of electron transfer on MgATP for the homologous complex.
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Electron Transfer Monitored by EPR Spectroscopy--
To confirm
that the increase in absorbance observed by UV-visible absorption
spectroscopy upon incubation of Cp2 with Av1 was caused by the
oxidation of the [4Fe-4S] cluster of Cp2, EPR spectroscopy was used
to monitor the oxidation state of the [4Fe-4S] cluster. The Cp2
[4Fe-4S]+ cluster is an S = 1/2 system
with a rhombic EPR spectrum centered at g = 1.93 (Fig.
2, trace 2) (33-35). Upon
oxidation to [4Fe-4S]2+, the cluster goes to an
S = 0 state and is EPR-silent (36). Dithionite-reduced
Av1 demonstrates low field EPR signals (g = 4.3 and
3.6) (not shown) and a signal at g = 2.0 (Fig. 2,
trace 1), which arise from FeMoco (37-39). The Fe protein
spectrum can be added to the MoFe protein spectrum to give a predicted
spectrum that would arise from a nonreacting mixture of Cp2 and Av1
(Fig. 2, trace 3) in dithionite. An EPR spectrum was
recorded for the mixture of Cp2 and Av1 after 1 h of incubation
(Fig. 2, trace 4). As shown, the intensity of the reduced
Cp2 signal centered at g = 1.93 decreased, consistent
with the one-electron oxidation of the [4Fe-4S] cluster of Cp2 to the
EPR-silent 2+ oxidation state. This result confirms the transfer of an
electron from Cp2 supposed from the changes in the absorbance
spectra.
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No changes were seen in the intensity or line shapes of the EPR signals
attributed to FeMoco of Av1 after incubation with Cp2. Likewise, EPR
signals ascribed to the P clusters did not appear upon incubation of
Av1 with Cp2. To confirm that Cp2 could transfer an electron to the P
cluster, which is the putative electron acceptor, changes in the EPR
signals of more oxidized states of the P clusters were monitored.
One-electron oxidation of the P cluster from the
PN state results in an S = 1/2
and 5/2 mixed spin state (designated as P+) that gives rise
to perpendicular mode EPR signals in the g = 2 and
g = 5 regions (40). Two-electron oxidation of the P
cluster from the PN state results in an
S 3 spin system (designated as P2+) that
gives rise to a parallel mode EPR signal at g = 11.8 (28). Therefore, it is possible to use EPR to monitor electron transfer from the Fe protein to the P cluster of the MoFe protein by monitoring the reduction of the P2+ oxidation state to the
P+ oxidation state (41). Fig.
3 (trace 1) shows the
perpendicular mode EPR spectrum of Av1 oxidized by four electrons (each
P cluster oxidized by two electrons to the P2+ state). The
existence of the P2+ oxidation state was evident by a
parallel mode EPR signal at g = 11.8 (data not shown).
The predicted EPR spectrum (obtained from the sum of spectra 1 and 2)
for the nonreacting mixture of Av1 in the P2+ oxidation
state and reduced Cp2 is shown in Fig. 3 (trace 3). When the
two proteins were actually allowed to react, however, the spectrum
shown in Fig. 3, trace 4, was obtained. The disappearance of
the g = 1.93 signal ascribed to Cp2 and the appearance
of signals with g values at 5.28, 2.04, 1.94, and 1.80 are
consistent with the oxidation of the [4Fe-4S]+ cluster to
the 2+ state and the reduction of each P cluster from the
P2+ to the P+ state. In addition, the parallel
mode EPR signal for P2+ disappeared after Cp2 and Av1 were
mixed (data not shown). In control incubations, when reduced,
dithionite-free Av2 was incubated with oxidized Av1 (P2+
state) in the absence of MgATP, no changes in the EPR spectrum were
observed, indicative of a lack of electron transfer in the absence of
nucleotides for the Av2-Av1 mixture.
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Rates of Primary Electron Transfer from Cp2 to Av1--
To obtain
kinetic constants for the observed electron transfer from Cp2 to Av1,
the changes in the electronic spectrum at 430 nm were monitored by
stopped-flow spectroscopy upon mixing reduced Cp2 with Av1 in the
absence of nucleotides. A first-order increase in the absorbance at 430 nm was recorded with an apparent first-order rate constant of 0.007 s1 (Fig. 4A,
trace 1). When MgADP was included, again an apparent first-order increase in the absorbance was observed, with an apparent first-order rate constant of 0.018 s
1 (Fig.
4A, trace 2). The addition of MgATP significantly
increased the rate of oxidation of Cp2 as evident in Fig.
4B. An apparent first-order rate constant for electron
transfer in the presence of MgATP was measured to be 100 s
1. This apparent rate constant for primary electron
transfer from Cp2 to Av1 in the presence of MgATP represents more than
a 104-fold increase from the nucleotide free condition and
approaches that observed for the MgATP-dependent rate
constants found for the homologous A. vinelandii nitrogenase
complex (42).
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Redox Titrations--
To gain insights into how nucleotides might
accelerate the rate of electron transfer in the Cp2·Av1 complex, the
midpoint potentials (Em) of the
[4Fe-4S]2+/+ couple of Cp2 and the
P2+/N couple of Av1 were determined for the
individual proteins alone and for the two proteins when complexed
together. According to electron transfer theory (43), the difference
between the Em values (or the free energy change
G) for the electron donor and the electron acceptor will
affect the rate of electron transfer (44). Em
values for the metal centers were also determined in the presence of
MgADP. Similar experiments in the presence of MgATP are not possible
because of MgATP hydrolysis by this nitrogenase complex. Fig.
5A presents redox titrations
for the [4Fe-4S]2+/+ cluster couple of Cp2 either in the
uncomplexed state or when complexed with Av1. From fits of the data to
the Nernst equation (Equation 1), Em values of
300 and
510 mV were determined in the absence of nucleotides for
the uncomplexed and complexed states, respectively. When MgADP was
included, the Em for the
[4Fe-4S]2+/+ couple of free Cp2 was shifted to
380 mV,
consistent with shifts seen earlier (45). When MgADP was added to Cp2
complexed with Av1, the calculated Em of the
[4Fe-4S]2+/+ cluster couple was
510 mV, the same as the
value calculated for the complexed Cp2 couple in the absence of
nucleotides. Fig. 5B presents redox titrations for the
P2+/N couple of Av1 in the uncomplexed state and
when complexed to Cp2, either in the absence of nucleotides or in the
presence of MgADP. Within the error of the experiment, no changes in
the Em for the P2+/N
couple were observed as a result of complex formation with Cp2 (approximately
310 mV). Likewise, the addition of MgADP to the Cp2·Av1 complex did not result in detectable changes in the
Em for the P clusters.
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DISCUSSION |
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The recent x-ray structures for two nitrogenase Fe protein-MoFe
protein complexes have provided important insights into the interactions between these two proteins (46, 47). However, some major
questions remain, including how the affinity between the two component
proteins is changed during the catalytic cycle, how MgATP binding and
hydrolysis are involved in this process, and how MgATP is coupled to
intercomponent electron transfer. Recent investigations of the three
known tight nitrogenase complexes, namely the Cp2·Av1 complex (18),
the MgADP·AlF4-stabilized Av2·Av1
complex (48), and the L127
Av2·Av1complex (49, 50), have begun to
provide some insights into these outstanding questions. For example,
the earlier observation that MgATP could be hydrolyzed at considerable
rates by the Cp2·Av1 complex (18) supported the idea that the Fe
protein and MoFe protein do not have to dissociate for exchange of
nucleotides to occur. Similar results have been found in the L127
Av2·Av1 complex.2 More
recently, examinations of the L127
Fe protein-MoFe protein tight
complex (49) and the
Av2·Av1·MgADP·AlF4
tight complex
(48) have revealed that primary electron transfer can occur within the
complex without MgATP hydrolysis. It was concluded, contrary to
longstanding models, that MgATP hydrolysis was not absolutely required
for electron transfer between the proteins. The present results for the
Cp2·Av1 nitrogenase complex further define a role for MgATP in the
electron transfer process by revealing that one important function is
to accelerate the primary electron transfer rate.
It is of interest to consider how nucleotides might function to
accelerate primary electron transfer within the complex. Perhaps the
simplest explanation would be that nucleotides change the thermodynamic
driving force for the electron transfer reaction. This could be
accomplished by nucleotide-induced changes in the Em values for either the Fe protein [4Fe-4S]
cluster or the MoFe protein P cluster, thereby increasing the
difference between the two Em values
(Em). This explanation is tempting
considering earlier work that demonstrated that MgATP or MgADP binding
to the free Fe protein results in about a
120 mV shift in the
Em for the [4Fe-4S]2+/+ couple
(10, 45). More recently, it has also been shown that association of the
L127
Fe protein with the MoFe protein to form a tight complex
results in shifts in the Em values of two of the three nitrogenase metal centers to favor electron transfer (50). The
Em for the L127
Fe protein
[4Fe-4S]2+/+ couple was observed to shift by more than
200 mV upon complex formation to about
600 mV, and the
Em for the MoFe protein
P2+/N couple shifted by
80 mV to
390 mV.
Negative shifts in the Em values of the P
cluster and the [4Fe-4S] cluster were also observed in the
Av2·Av1·MgADP·AlF4
nitrogenase
complex (48). In the present work, the Em values for the metal centers of free Cp2 and free Av1 and for the Cp2·Av1 complex were determined. Em values observed for
the free proteins were consistent with previously reported values (28,
45, 51). In the complex, only the Em of the
[4Fe-4S] cluster changed (from
300 to
510 mV). Unlike the other
two complexes described above, no change in the
Em of the P cluster was observed in the
Cp2·Av1 complex. Despite the particular differences that complex
formation has on the Em values for each metal
cluster in the three tight complexes, a theme that emerges is that the
energy of complex formation is coupled to changes in the
Em values to favor electron transfer.
Although MgADP is observed to accelerate the electron transfer rate in the Cp2·Av1 complex by 2.5-fold, MgADP resulted in no additional changes in the Em values for any of the metal centers within the complex. This observation suggests that the rate acceleration induced by MgADP is not the result of changes in the driving force (i.e. Em values). Although it is not possible to measure the effects of MgATP on the Em values in the complex because of the hydrolysis reaction, electron transfer theory can be used to make the argument that, similar to MgADP, the rate acceleration in electron transfer induced by MgATP cannot be accounted for solely by changes in driving force. It should be noted that the electron transfer reactions in the Cp2·Av1 complex may not necessarily follow electron transfer theory, but for the purposes of this argument this assumption will be made. A simplified version of the Marcus equation which has evolved from analysis of many protein electron transfer reactions (44) is shown in Equation 3
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(Eq. 3) |
Another possible way to increase the rate of electron transfer could be through changes in the pathway(s) for electron transfer. Changes in electron transfer pathways in other proteins have been observed to have large effects on the electron transfer rate (54). Possible electron transfer pathways from the Fe protein [4Fe-4S] cluster to the MoFe protein P cluster can be suggested from the structures of the two nitrogenase complexes (46, 47). In the two likely pathways, at least one H-bond (i.e. noncovalent bond) jump is predicted. Such jumps have been suggested to be less efficient in electron transfer (55, 56). One possibility is that MgATP binding or hydrolysis in the Cp2·Av1 complex could result in changes in the electron transfer pathway, possibly including changes in the distance or orientation of the H-bond jump. In this case, some of the energy associated with MgATP binding or hydrolysis would be coupled to changes in protein conformation within the complex that would alter the electron transfer pathway.
Finally, two additional observations from the present work deserve
comment as they provide additional insights into the roles of MgATP in
the nitrogenase reaction. First, it is evident that for the Cp2·Av1
nitrogenase complex, the acceleration of the electron transfer rate is
at least partially uncoupled from the rates of MgATP hydrolysis.
Although MgATP addition accelerated the primary electron transfer to
rates near those observed for the homologous nitrogenase complexes, the
rates of MgATP hydrolysis for the Cp2·Av1 complex are 36-fold lower
than for the homologous complex (18). It has been known for some time
that MgATP hydrolysis can occur in nitrogenase complexes without
electron transfer, thus indicating that hydrolysis can be uncoupled
from electron transfer. The present results indicate that electron
transfer rates can also be uncoupled from MgATP hydrolysis rates. This
latter point is supported by the observations of electron transfer
without MgATP in other nitrogenase tight complexes (48, 49). Finally,
an important observation from the present work is that Cp2 is unable to
transfer more than one electron into the resting state of the MoFe
protein even when MgATP is present. After electron transfer, the
[4Fe-4S] cluster of Cp2 can be reduced in the complex, yet no further
electron transfer (i.e. oxidation of the [4Fe-4S] cluster)
is observed. This would explain why the Cp2·Av1 complex is inactive
because at least two electrons would be required to reduce even the
simplest substrates (57). The reason that the second electron cannot be
transferred in the Cp2·Av1 complex is not clear. It would appear from
the current results that the limitation is not the hydrolysis of MgATP
because MgATP hydrolysis continues in the Cp2·Av1 complex before and
after the primary electron transfer event. Instead, the lack of
transfer of the second electron into the MoFe protein may have to do
with the proper coupling of MgATP binding or hydrolysis to changes in
the Fe protein or MoFe protein structures, or the oxidation state of
the electron acceptors in the MoFe protein. The lower rates of MgATP
hydrolysis by the Cp2·Av1 complex may also account for the lack of
transfer of a second electron. Additionally, dissociation of the Fe
protein from the MoFe protein, which does not occur readily in the
Cp2·Av1 complex, may somehow be required for subsequent electron
transfer reactions. Studies to begin to understand these open questions
are currently under way.
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ACKNOWLEDGEMENTS |
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We thank Drs. William Lanzilotta, Jason Christiansen and Jennifer Huyett for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-9722937 (to L. C. S.) and by a Willard L. Eccles Foundation fellowship (to J. M. C).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 435-797-3964;
Fax: 435-797-3390; E-mail: seefeldt{at}cc.usu.edu.
2 M. J. Ryle, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
MoFe protein, molybdenum-iron protein of nitrogenase;
Fe protein, iron protein of
nitrogenase;
FeMoco, [Fe-Mo-9S-homocitrate[ cofactors;
Av1, molybdenum-iron protein from A. vinelandii;
Av2, iron
protein from A. vinelandii;
Cp2, iron protein from C. pasteurianum;
MOPS, 3-(N-morpholino)propanesulfonic
acid;
Tricine N-tris(hydroxymethyl)methylglycine, Em, midpoint potential;
P cluster, [8Fe-7S]
cluster of MoFe protein;
Pn, oxidation state of
the P cluster with all ferrous atoms;
P+, P cluster
one-electron oxidized from the Pn state;
P2+, P cluster two-electron oxidized from the
Pn state;
G, free energy
change.
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REFERENCES |
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