Chapter 19
Electron-Transfer Reactions Associated with Nitrogenase from Klebsiella pneumoniae
Molybdenum Enzymes, Cofactors, and Model Systems Downloaded from pubs.acs.org by YORK UNIV on 12/07/18. For personal use only.
R. N. F. Thorneley, G. A. Ashby, K. Fisher, and D. J. Lowe Agricultural and Food Research Council, Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton BN1 9RQ, United Kingdom
Pyruvate supported nitrogen fixation in Klebsiella pneumoniae principally involves four proteins: a pyruvate-flavodoxin oxidoreductase (nifJ protein); a flavodoxin (KpFld); the nitrogenase Fe-protein (Kp2); and the nitrogenase MoFe-protein (Kp1). Recent advances in understanding this electron transfer pathway are reviewed These include: a novel post-translational modification of KpFld by covalent attachment of coenzyme A that may constitute part of a new regulatory mechanism for nitrogen fixation; the demonstration that the rate constants that define the Fe-protein cycle of nitrogenase are independent of the redox level of Kp1; the effect of changing ß-Phe124 ofKp1to Met orIleon MgATP-dependent electron transfer from Kp2; and the kinetic analysis of 'slow' absorbance changes and EPR signals associated with Kp1 during substrate reduction that indicate a key role for the 'P'-centers. This article reviews recent advances made by the Sussex Group and collaborating laboratories in understanding the mechanism of action of the Mo-containing nitrogenase of Klebsiella pneumoniae and its associated electron transfer pathway. A detailed description of the structures of the two proteins that comprise Mocontaining nitrogenases is given elsewhere in this volume(Burgess, chapter 10; Rees et ai, chapter 11; Bolin, chapter 12) and in the recent reviews by Thorneley (/), Eady (2), Lowe (5), and Smith and Eady (4). Scheme 1 shows the electron transport chain from pyruvate to nitrogenase in K. pneumoniae. The sections below detail sequentially in the direction of electron movement the effects of a novel post-translational modification of the nifF gene product, a flavodoxin (KpFld), by coenzyme A (5); the effect the first one-electron reduction of the MoFe-protein has on reactions occurring in the second Fe-protein cycle (6); the involvement of Phel24 on the β-chain of the MoFe-protein in MgATP-dependent electron transfer from the Fe protein (7); and a role for the 'P'-centers in reducing dinitrogen (8). 0097-6156/93/0535-0290$06.00/0 © 1993 American Chemical Society
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Nif J
Nif F
NifH
NifD NifK
PYRUVATE FLAVODOXIN bXIDOREDUCTASE
FLAVODOXIN
FePROTEIN
MoFePROTEIN
CoA Pyruvate Acetyl CoA CO_
2ATP
2ADP + 2Pi
Scheme 1. Electron Transport Chain to Nitrogenase in K. pneumoniae. Post-translational Modification of K. pneumoniae Flavodoxin by Coenzyme A Flavodoxins are small (M, « 20 K) flavin-mononucleotide (FMN)-containing, monomeric proteins that act as low potential (E^ in the range -100 to -520 mV NHE for the physiologically important semiquinone-hydroquinone couple), single electron donors to nitrogenase. The electron transport chainfrompyruvate to nitrogenase that involves a flavodoxin (the nifF gene product, KpHd) in K. pneumoniae is shown in Scheme 1. The expression of the nifF gene in diazotrophs is repressed by ammonia (when nitrogenase activity is not required for growth) (9) or in the presence of excess oxygen when nitrogenase is irreversibly inactivated (10). However little is known about the regulation of K. pneumoniae nitrogenase activity in vivo. There is no evidence for an ADP-ribosylation system in K. pneumoniae such as that which operates in Rhodospirillum rubrum to render nitrogenase Fe-protein inactive when cultures are exposed to high ammonia concentrations (11). However, chemostat studies have shown that nitrogenase activity of K. pneumoniae can be rapidly 'switched off and on' in response to the concentration of dissolved 0 in the medium (70). The novel post-translational attachment of coenzyme A to KpFld described below may be part of this regulatory mechanism acting as an 'aerobic-anaerobic' switch for nitrogenase activity. The nifF gene product (KpFld) is present at very low concentrations in K. pneumoniae even when growth is under N -fixing conditions. Hence for kinetic studies with nitrogenase and crystallization trials it has been over-expressed in Escherichia coli so that it comprises «15% of the soluble protein (5). Two forms of KpFld were separated by f.p.l.c. Since both these proteins had the same N terminal amino acid sequence (30residues)that correspond to that predicted from the nifF DNA sequence (72), a post-translational modification was suspected. The P NMR spectra in Figure 1 showed that the modified form of KpFld exhibited an additional singlet peak and a multiplet (AB quartet). The assignment of these P-resonances to the phosphate groups of coenzyme A was made after highly accurate M , values were obtained for the two forms of KpFld using the recently developed technique of electrospray mass spectrometry (Table I). The value of 18984 for KpFld is very close to that of 18981 calculated from the DNA-derived 2
2
31
3l
292
MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS II H
c
ο
H—
c I
OH
Η—
P O j
2-
fa
C—OH
Η—C—OH
FMN
I
Coenzyme A
Figure 1. Post-translational modification of K. pneumoniae flavodoxin by covalent, mixed disulfide attachment of coenzyme A. Phosphate groups designated A, Β and C give rise to the correspondingly labelled P NMR peaks. 31
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293
amino acid sequence assuming the dissociation of non-covalently bound FMN cofactorfromthe peptide in the electrospray apparatus. The difference in the Mr for the two forms of KpFld allowed us to assign coenzyme A as the most likely modifying group. The measured increase in Mr of 765.6 is remarkably close to the M , of coenzyme A (767.5 for CoA-SH) and in exact agreement if coenzyme A is attached to KpFld by a mixed disulfide bond formed by the loss of two protons. The linkage was confirmed by thiol group estimations and by 'chemical synthesis' of the modified form by incubation of KpFld with coenzyme A. The modifying group was confirmed as coenzyme A by removing it from KpFld by incubation with dithiothreitol and then assaying this using the coenzyme A specific enzyme, acetylCoA:orthophosphate acetyl transferase.
Table I. Electrospray Mass Spectrometry of Post-translationally Modified K. pneumoniae flavodoxin Molecular Weight Calculated
Observed
KpFld
18981.4
18984.0 ± 3.8 18984.2 ± 3.5
KpFld-CoA
19747.9
19748.7 ± 1.7
KpFld-CoA*
19747.9
19754.0 ± 7.1
Observed Average Difference
765.6 ±35
The data were obtained with 10 μΐ, 150 pmol protein samples injected into a VG BIO-Q quadrupole mass spectrometer. * Synthesized by incubation of KpFld with coenzyme A. Since KpFld only contains a single cysteine residue (position 68), this must be the site of coenzyme A attachment to the protein. The x-ray structure of homologous flavodoxin from Azotobacter chroococcum (AcFldB) shows that this residue is located ca. 5Âfromthe FMN cofactor, Figure 1 (P.M. Harrison, G. Ford and A. Shaw, personal communication). Not surprisingly, the coenzyme A modified flavodoxin is unable to act as an electron carrier between the nifJ protein and the nitrogenase Fe-protein, Figure 2 (5). Interestingly, only the electron transferfromthe nifJ gene product to theflavodoxinsemiquinone is prevented by the coenzyme A sited, adjacent to the edge of the FMN, on the surface of the protein. Electron transferfromdithionite-reducedflavodoxinhydroquinone to oxidized nitrogenase Feprotein does not appear to be affected by the presence of the coenzyme A. An attractive explanation is that the presence of coenzyme A on KpFld stabilizes the protein complex formed with nifJ. The coenzyme A attached to KpHd-Cys68 could occupy the site on the nifJ protein that is normally involved in the conversion of
294
MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS
coenzyme A to acetyl-coenzyme A during oxidation of pyruvate, i.e., KpFld-CoA is a competitive inhibitor of coenzyme A binding (as a substrate) to nifJ protein. 100 Γ
[KpFld] (μΜ) Figure 2, Pyruvate-flavodoxin oxidorcductase-supported acetylene reduction, catalyzed by nitrogenase, is mediated by native KpFld but not by CoA-modified KpFld. See ref. (5) for reaction conditions. The competent electron transfer to nitrogenase Fe-protein could imply that electrons can leave KpFld-CoA at a site other than the exposed edge of the FMN. Drummond (13) has commented on the pronounced dipole moment of KpFld. He noted five basic residues on helix 1, densely clustered on the surface nearest the FMN and seven basic residues on the surface of helix 2 and the segment immediately before it. The high resolution structures of Nif-specific flavodoxins and nitrogenase Fe proteins that are now becoming available should enable meaningful computer modelling of a physiologically functional electron transfer protein complex. In particular, the effects changes in the redox states of both proteins and of nucleotideinduced conformation changes of the Fe-protein have on the structure/function of the electron transfer protein-complex are intriguing (14). Coenzyme A levels in vivo are likely to be sensitive to the 0 status of the cell and this novel coenzyme A dependent post-translational modification of flavodoxin must be a strong candidate for the sought after 'aerobic-anaerobic switch' that regulates nitrogenase activity. 2
Reduction of the MoFe Protein Does Not Change the Kinetics of the Next Fe Protein Cycle Our current understanding of the complex kinetics and mechanism of nitrogenase is based on five papers that describe the partial reactions that define the Fe-protein cycle (15) and the subsequent combining of eight of these Fe-protein cycles into a single MoFe-protein cycle in which N is reduced and H evolved (16-19). It is possible to restrict the system to the first two cycles only by working at low electron 2
2
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Electron-Transfer Reactions
THORNELEY ET AL.
flux through the MoFe-protein (Fe-protein/MoFe-protein ratio of 1:100). Under these conditions, only H is evolved since the electron flux is not sufficient to generate a significant concentration of the N -binding forms of the MoFe-protein (Ej and E in Scheme 1 of ref. 16). Thus a subcycle involving only MoFe-protein in states EQ, E J and Ej can describe the system (Scheme 2). 2
2
4
4-7
E H • • E H , etc. 2 2
3
3'
EH 1
Scheme 2. MoFe-protein Cycle for H Evolution at Low Electron Flux. EQ represents one of two independently functioning halves of the tetrameric (ajijj) structure of the K. pneumoniae MoFe-protein. The subscript refers to the number of times the electron transfer Fe-protein cycle has been completed and therefore represents the number of electron equivalents by which the αβ MoFeprotein moiety has been reduced relative to resting Eo. Species EQ has an EPR signal (g-values 4.3, 3.7, 2.01) while species EjH and E ^ are EPR silent. The rate constants for the Fe-protein cycle are defined in reference (16). 2
A key assumption of our scheme for the mechanism of nitrogenase action (16) is that the rate constants of the Fe-protein cycle are not dependent on the reduction level of the MoFe-protein. If this assumption is correct, then Scheme 2 predicts that, a) the EPR signal of the MoFe-protein should be 50% bleached when a steady-state has been achieved after a lag phase of several minutes, b) the kinetics of the bleaching of the EPR signal should parallel the lag phase for evolution and c) stopped-flow absorbance changes associated with MgATP dependent electron transfer from Fe- to MoFe-protein, induced by mixing MoFe-protein with an excess of Fe-protein, should be independent of the redox level of the MoFe-protein. We have verified these predictions and shown that the rates of complex formation (k = 5xl0 M* s" ), electron transfer (k^ with a k ^ = 140 s' , Figure 3) and oxidized-complex dissociation (k_ = 6.4 s" ) are the same for Kpl in states EQ (first Fe-protein cycle) and E H (second Fe-protein cycle) (6). 7
x
1
1
3
t
1
1
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MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS
The demonstration that EQ and E are reduced by the Fe-protein at the same rate suggests that either electron transfer per se is not rate limiting or that the site on the MoFe-protein receiving the electron is at the same redox level in EQ and E ^ The first possibility is consistent with ATP-hydrolysis or a protein conformation change being rate limiting; the second with electron transfer occurring to a site other than the FeMo-cofactor, the E P R signal of which is ultimately bleached. The *P' centers are obvious candidates to initially receive the electron from the Fe-protein (see below). The same rate of complex formation for Fe-protein with MoFe-protein in states EQ and E indicates that reduction of MoFe-protein does not lead to a significant change in protein conformation or charge density at the surface that docks with the Fe-protein. This would be consistent with the neutralization by protonation of the additional negative charge generated on the FeMo-cofactor by reduction to yield metal hydrides as precursors to H evolution and N binding. X
T
2
10 20 Time (ms)
2
30
10
20 Time (ms)
30
Figure 3. MgATP induced electron transfer from Fe-protein to MoFe-protein at oxidation levels EQ and EjH occurs at the same rate. Trace (a) is for 100% EQ and trace (b) for 50% EQ and 50% E ^ . Traces (a) and (b) are essentially identical and are bestfittedby a single exponential function (k^ = 140 s" ). The dotted and broken lines in trace (b) (which do not fit the data as well as the solid line) were drawn assuming that EQ and E H are reduced at different rates. See ref. (6) for further details. 1
t
P-Phel24 of K. pneumoniae MoFe-protein Implicated in MgATP-Dependent Electron Transfer from the Fe-protein The K. pneumoniae MoFe-protein has a single chymotrypsin cleavage site at Phel24 on the β-subunit in a highly conserved region between Cys94 and Cysl52 (7). This site must be accessible to chymotrypsin and is therefore on, or close to, the surface of the β-subunit. As such, it could be part of a "docking-site" for binding the Feprotein. Since MgATP dependent electron transfer only occurs within the Fe-MoFeprotein complex, kinetic parameters associated with these partial reactions might be expected to change if Phel24 is changed to another residue by site specific mutagenesis. Subsequent to the results described below being obtained, it has been
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Electron-Transfer Reactions
confirmed that Phel24 is on the surface and that the adjacent Cys94 provides a thiolate ligand to bridge the two 4Fe-4S clusters that comprise each 'P'-center (assuming structural homology between Kpl and A v l , Rees et al., Chapter 11). Two recombinant Kpl proteins, p-Phel24Ile and P-Phel24Met, have been purified with similar Mo and Fe contents to native K p l . As expected, both proteins were totally resistant to proteolysis by chymotrypsin. The p-Phel24De protein had a lower specific activity than either |i-Phel24Met or native K p l , Table II.
Table IL Kinetic Effects of Changing β-ΡΗβ124 to Met and De $-Phel24
$-Phel24Ik
$-Phel24Met
Electron transfer Us" )
140
130
40
MgATP binding Κο(μΜ)
400
320
110
Spec, activity per ng atom Mo
283
259
107
1
Stopped-flow spectrophotometry showed that MgATP dependent electron transfer from Fe-protein (Kp2) to P-Phel24De was much slower (1^= 40 s") than to p-Phel24Met (1^= 130 s") or native Kpl (1^= 140 s" ), Figure 4 traces (a) and (c). The dependences of on MgATP concentration showed that the P-Phel24IleKp2 protein complex binds MgATP (Ku = 110 μΜ ) significantly tighter than do the p-Phel24Met- and native Kpl-Kp2 protein complexes (K^ = 320 μΜ and 400 μΜ respectively). Stopped-flow absorbance changes at times between 0.1 and 1.0 s (Figure 4 traces (b) and (c)) were quite different for the P-Phel24Ile than for βPhel24Met or native protein. Instead of the characteristic "slow-effects" shown in trace (b) (discussed in detail below), the absorbance in trace (d) decreases essentially to the initial value. This means that in the steady state a much higher percentage of the Fe-protein is in the reduced state. If complex dissociation (k_ in the Fe-protein cycle) remains as the rate-limiting step, then k. must decrease from 6.4 s" to ca. 2.4 s" to be consistent with the lower specific activity of |i-Phel24Ile (Table Π). However a decrease in k_ should result in a higher steady-state concentration of oxidised Fe-protein, not the observed decrease. We conclude therefore that with Phel24Ile another reaction has become rate limiting and that this effectively slows down the oxidation of the Fe-protein in the second cycle. Since the Phel24Ile mutation effects MgATP binding to the protein complex, we speculate that this may involve energy transduction and electron transfer occurring within the MoFe-protein involving a 'P'-center that is now known to be adjacent to Phel24 (Rees et al, Chapter 11). 1
1
1
3
1
3
1
3
1
Figure 4. MgATP induced electron transfer from K. pneumoniae Fe-protein to P-Phel24Met and |$-Phel24Ile. The traces were obtained by stopped-flow spectrophotometry at 430 nm when pre-equilibrated nitrogenase component proteins Kp2 (80 μΜ) with P-Phel24Met (traces a and b) or p-Phel24Ile (20 μΜ) (traces c and d) were mixed with MgATP in the presence of sodium dithionite. The single exponentials drawn through traces (a) and (c) give the first order rate constants shown in table Π. Trace (d) shows a single exponential absorbance decrease (k^ = 3 s") occurring after primary electron transfer.
r S§ Η 3
§
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A Role for 'P'-Centers in Nitrogen Reduction Previous stopped-flow spectrophotometric studies of the pre-steady-state phases of nitrogenase have utilised absorbance changes associated with the oxidation/reduction of the Fe-protein and have not considered, in any detail, changes due to the MoFeprotein (6,15,20,21). This is because the technique has been used to determine the rate constants that describe the partial reactions of the first (15) and second (6) Feprotein cycles. We have previously not attempted to explain or simulate the small, kinetically complex absorbance changes, occurring at times up to 1 s (the so called 'slow effects'), simply because we did not understand them. Recendy (S), by combining 'slow effect' data, obtained with stopped-flow spectrophotometry under different gaseous, reducible substrates (Ar{H }, N , and with EPR data obtained under the same conditions, we have been able to rationalize and simulate these effects, Figure 5. These simulations used the Lowe-Thorneley model for H " (76), N (17) and C f t (22) reduction, the published rate constants for the partial reactions (18), and assigned Δε values to the MoFe-protein as it is reduced sequentially from state EQ through E and to the N binding states E and E , Table HI. The most significant conclusion is that the reductive formation of E is associated with the oxidation of a cluster within the MoFe-protein, and furthermore, by EPR, that the centers oxidized are the 'P'-centers, Figure 6. Thus, at E electron transfer from the P'-centers to FeMoco is triggered resulting in increased reducing power at the N -binding/reduction site. We have previously shown that an irreversible step at E , after N has bound, is necessary in order to simulate the dependence of the for N on [Kp2]:[Kpl] and suggested that this step was a protonation of bound N to yield a hydrazido(2') intermediate. +
2
1
2
430ηιη
t
2
3
4
4
4
4
2
4
2
2
2
Table ΙΠ. Extinction Coefficient Changes Associated with MoFe-protein (Kpl) During the Pre-steady-state Phase of Substrate Reduction 1
Δ ε ^ (mM'cm )
Reaction
Comment
