Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Available online at www.sciencedirect.com JOURNAL OF

Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 1649–1656 www.elsevier.com/locate/jinorgbio

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function Karl Fisher a

a,b

, David J. Lowe b, Pedro Tavares c,1, Alice S. Pereira Dale Edmondson d, William E. Newton a,*

c,1

, Boi Hanh Huynh c,

Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA b Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK c Department of Physics, Emory University, Atlanta, GA 30322, USA d Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Received 26 April 2007; received in revised form 10 July 2007; accepted 13 July 2007 Available online 9 August 2007

This manuscript is dedicated to the memory of Ed Stiefel, a fine scientist, a good friend and an excellent colleague, who always sought and relished a new frontier

Abstract Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the aH195Q, aH195N, and aQ191K variant MoFe proteins (where His at position 195 in the a subunit is replaced by Gln/Asn or Gln at position a-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N2 with the aH195Q and aH195N variants, but not with the aQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, aH195Q or aH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the aQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze 57Fe Mo¨ssbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, aH195Q and aH195N MoFe proteins can bind N2, but aQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N2 reduction. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Nitrogen fixation; Nitrogenase; Azotobacter vinelandii; Rapid-freeze EPR spectroscopy; Rapid-freeze Mossbauer spectroscopy; Stopped-flow spectrometry; P cluster; FeMo-cofactor

1. Introduction *

Corresponding author. Tel.: +1 540 231 8431; fax: +1 540 231 9070. E-mail address: [email protected] (W.E. Newton). 1 Present address: REQUIMTE/CQFB, Departamento de Quimica, Faculdade de Cieˆncia e Tecnologia, Universidade Nova de Lisboa, 2829516 Caparica, Portugal. 0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.07.037

Molybdenum-dependent nitrogenase (Mo-nitrogenase) from Azotobacter vinelandii is composed of two metalloproteins, the MoFe protein and the Fe protein [1]. Crystal structures of the individual proteins and their complex

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[2–8] have defined the disposition and structure of the two types of prosthetic groups, the FeMo-cofactors and P clusters, accommodated within the MoFe protein. Although these resting-state structures are important in the design of mechanistic probes, they do not necessarily provide insight into the catalytic forms of the enzyme, which may result from substantial redox and structural changes. Furthermore, the redox states of the clusters within the crystallized proteins are almost always unknown. The FeMo-cofactor [9] has the composition, [Mo–7Fe–9S–X– homocitrate] (where X is an as-yet unidentified small atom or ion within the central cavity; [10–12]), and overwhelming evidence indicates that it contains the sites which bind substrates and inhibitors [13–15]. The P cluster has the composition, [8Fe–7S]; however, its role in nitrogenase catalysis remains unclear. On complex formation, the MoFe protein receives electrons, one-at-a-time when dithionite is used as the reductant, from the Fe protein [16–18]. These electrons are generally assumed to be accepted by the P clusters, which then forwards them to the substrate via the FeMo-cofactor. Each such Fe protein–MoFe protein association and electron transfer event involves hydrolysis of both MgATP molecules bound to the Fe protein. Dissociation of the complex then occurs to allow the now oxidized Fe protein to be re-reduced and to exchange the spent nucleotide in order to re-enter the catalytic cycle [19,20]. However, Mo¨ssbauer spectroscopy has shown that all 8 Fe atoms of the P cluster are ferrous in the resting-state MoFe protein [18–21]. This situation then raises the question as to how such a cluster is able to accept additional electrons from the Fe protein during enzyme turnover. Various spectroscopies have played important roles in elucidating the structure and properties of the nitrogenase metal clusters. An important probe of the reactions of the MoFe protein is the rhombic EPR spectrum of its resting state, which has g-values of 4.3, 3.7 and 2.0 and originates from the FeMo-cofactor [22–24]. Electron transfer from the Fe protein to the MoFe protein results in the bleaching of the EPR spectra of both the Fe protein and the MoFe protein as the proteins become oxidized and reduced, respectively. Multiple variations of this S = 3/2 signal have been observed for the MoFe protein from Klebsiella pneumoniae [25] and for some variant A. vinelandii MoFe proteins that contain specific substitutions in the FeMo-cofactor environment [26,27]. These latter signals have been suggested to represent either trapped turnover intermediates or FeMo-cofactors bound in different conformations. Various other EPR signals associated with the MoFe protein are observed in the presence of either CO [28–30] or C2H2 and C2H4 [25]. Most of the MoFe protein redox states produced during nitrogenase turnover are unlikely to be accessible by crystallography. We have, therefore, used rapid-mixing/rapidfreezing techniques and EPR spectroscopy to explore changes both in the electronic structure of the MoFe protein and in the conformation/protonation state of the

FeMo-cofactor that occur during the pre-steady-state phases of turnover [31]. We found that rapid-freezing of a turning-over 3:1 molar ratio mixture of wild-type A. vinelandii Fe protein and MoFe protein elicited two transient EPR signals (signals 1b and 1c) from the FeMo-cofactor within 500 ms after mixing. The first signal (called 1b with g = 4.21, 3.76) observed was formed at the expense of the resting-state signal, whereas the second signal (called 1c with g = 4.7, 3.4) formed more slowly and in lower intensity. The loss of signal 1a intensity was never fully compensated by those of signals 1b plus 1c, suggesting the presence of other EPR-silent species. Both signals formed under a N2, C2H2, or argon atmosphere. Simulations of the kinetics of signal 1b formation indicated that it arose after three electrons had been transferred to the MoFe protein. EPR signal 1b also closely resembled the EPR signal reported for K. pneumoniae MoFe protein at high pH [24], which presumably reflects deprotonation and a changed conformation around the FeMo-cofactor. In the current work, we sought to determine whether these presumed deprotonation and conformational changes were a reflection of catalytic capacity. To this end, we subjected three variant MoFe proteins, the aH195Q, aH195N, and aQ191K MoFe proteins (where His at position 195 in the a subunit was replaced by Gln/Asn or Gln at position a-191 by Lys) to a similar study. All three variants exhibit a resting-state S = 3/2 EPR signal but each is compromised in one or more important aspects of catalysis. Would the EPR spectrum of each of these three variants respond similarly to wild-type during the first 500 ms or so after turnover was initiated? We also extended our studies to include stopped-flow spectrophotometry and rapid-freeze Mo¨ssbauer spectroscopy to explore any change in the electronic structure of the P cluster during the pre-steady-state phases of nitrogenase turnover. 2. Materials and methods 2.1. Cell growth and protein purification The growth of wild-type (a-191Gln/a-195His), DJ255 (a191Lys/a-195His), DJ178 (a-191Gln/a-195Asn), and DJ540 (a-191Gln/a-195Gln) strains of A. vinelandii, nitrogenase derepression, cell-extract preparation, purification of the nitrogenase MoFe protein component, and exchange into 25 mM HEPES (pH 7.4) were performed as previously described [32–34]. The resulting MoFe protein specific activities were 2600 (wild-type), 1500 (aQ191K), 1500 (aH195N), and 2700 (aH195Q) nmol H2 (min mg MoFe protein) 1, under 101 kPa Ar in the presence of a 20-fold molar excess of wild-type Fe protein with an ATP/2e value of 5.1 ± 0.5. MoFe protein was labeled with 57Fe for Mo¨ssbauer studies by growing wild-type A. vinelandii cells by the same procedure as above except that 57Fe-enriched metal (95% plus enrichment from Advanced Materials and Technology, New York, NY) replaced the usual iron source. The 57 Fe-labeled MoFe proteins had a specific activity of 3000

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(wild-type), 1500 (aQ191K), 1400 (aH195N), and 2800 (aH195Q) nmol H2 (min mg protein) 1. The Fe-protein samples used in these experiments had a specific activity of 2300–2800 nmol H2 produced (min mg) 1. The method of Lowry et al. [35] was used for protein-concentration determinations and SDS-PAGE with Coomassie Blue staining was used to determine the state of homogeneity of all proteins. All buffers were saturated with argon and contained 2 mM sodium dithionite. Metal content was measured by inductively-coupled plasma atomic emission spectroscopy on a Perkin–Elmer Plasma 400 spectrometer. The molybdenum content was 0.9 Mo atoms per molecule for both the aQ191K and aH195N MoFe proteins and 1.9 Mo atoms per molecule for the wild-type and aH195Q MoFe proteins. The iron-to-molybdenum ratio was, however, constant at 13.5 ± 0.5:1 for all MoFe proteins, indicating an unchanged ratio of FeMo-cofactor-to-P cluster in all four MoFe proteins. 2.2. Nitrogenase assays Assays were performed at a total protein concentration of 0.5 mg mL 1 with a 20-fold molar ratio of wild-type Fe protein over either wild-type or variant MoFe protein at 30 °C in 9.25 mL reaction vials fitted with butyl rubber stoppers and crimped with aluminum caps as described previously [31]. Dihydrogen evolution was measured by gas chromatography on a molecular sieve 5A column (Supelco, Bellefonte, PA) using a thermal conductivity detector and calibration by a standard gas mixture of 1% H2 in N2 (Scott Specialty Gases Inc., Plumsteadville, PA). Creatine, as a measure of MgATP hydrolysis, was determined by the method of Ennor [36]. 2.3. Rapid-freeze sample preparation for EPR and Mo¨ssbauer investigations The apparatus used was essentially as described earlier [31,37]. An electronically controlled stepping motor expels a pre-set volume of solution from each reaction syringe at a pre-determined rate. The solutions travel through a mixing chamber and down a length of thick-walled nylon capillary tubing of variable length ending in either a needle jet or a spray nozzle. The length of this capillary tubing determines the time the reaction is allowed to proceed before quenching takes place. All reaction tubes were cleaned and then flushed with N2 before each shot. The needle jet was situated immediately above a glass funnel attached to either a quartz EPR tube or a Mo¨ssbauer cuvette, which were seated in an iso-pentane bath cooled to 140 °C with liquid N2. After freezing, the reaction mix was packed tightly into the bottom of the tube or cuvette with a PTFE-tipped packing rod. The samples were stored in liquid N2 until analyzed. EPR spectra were recorded on the day of preparation. Unless otherwise stated, one of the reaction syringes contained MoFe protein and Fe protein at the desired molar ratio and the second contained 18 mM ATP and

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20 mM sodium dithionite. All solutions contained 25 mM HEPES buffer (pH 7.4) and 10 mM MgCl2 and were saturated with N2. 2.4. EPR spectroscopy X-band frequency EPR spectra were recorded on a Bruker ESP300 spectrometer operated in perpendicular mode and equipped with an ESR 900 continuous-flow helium cryostat (Oxford Instruments). The magnetic field and the microwave frequency were determined with a NMR gaussmeter and a microwave counter. Spectra were recorded at 10 ± 0.2 K with a microwave power of 100 mW and a 100-kHz field modulation of 2 mT. To estimate the uncertainty in EPR-signal intensity introduced by differences in rapid-freeze packing and running-temperature fluctuations, replicate samples, in identical tubes and under constant He-flow conditions, gave EPR signals with intensities within ±10% of each other. The concentrations of intermediates were estimated from comparative peak heights and are expressed as a percentage of the starting intensity of the resting MoFe protein EPR signal, unless otherwise stated. 2.5. Stopped-flow spectrophotometry Stopped-flow spectrophotometry was used to measure the rates of primary electron transfer from the Fe protein to the MoFe protein [38] and to detect the relatively slow oxidation at longer times [39]. Stopped-flow spectrophotometry was performed using a commercially available SF-61 instrument equipped with a kinetic data acquisition analysis and curve-fitting system (Hi-Tech, Salisbury, Wiltshire, UK). The SHU–61 sample handling unit was installed inside an anaerobic chamber (Vacuum Atmos. Corp., Rosedale, CA) operating at <1 ppm O2. Sample flow components were thermostated by closed circulation of water through a Techne C-85D circulator (Techne Ltd., Duxford, Cambridge, UK) attached to a FC-200 Techne flow cooler situated outside the anaerobic chamber. All stopped-flow reactions were conducted at 23 °C in 25 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2 and 50 mM NaCl. For all determinations, one syringe contained a mixture of the MoFe protein (20 lM) and Fe protein in the appropriate molar ratio plus 10 mM Na2S2O4, whereas the other syringe contained 20 mM MgATP and 10 mM Na2S2O4. All reactions were run for at least 2 s. 2.6. Mo¨ssbauer spectroscopy Mo¨ssbauer spectra were recorded at 4.2 K in a 50-mT magnetic field applied parallel to the c-radiation by using a previously described spectrometer [37]. Theoretical spectra of the FeMo-cofactor and P cluster were simulated using the program WMOSS (WEB Research) with parameters previously reported for these clusters [21,40,41]. These theoretical spectra were then used to

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estimate the percent absorptions of these clusters in each experimental spectrum. 3. Results 3.1. New EPR signals detected by rapid-freeze techniques Fig. 1 shows typical EPR spectra obtained for the aH195Q MoFe protein in 3:1 molar ratio mixture with wild-type Fe protein at various times after initiating turnover under either a N2 or argon atmosphere. Similar spectra were obtained with the aH195 N MoFe protein (data not shown). As found for wild-type [31], two new signals were elicited, corresponding to signals 1b and 1c, at the expense of the original EPR signal 1a. In contrast to wild-type, signals 1b and 1c appeared together, arose quicker, and reached equilibrium sooner (by 350 ms) with both the aH195Q (Fig. 2) and aH195N (data not shown) MoFe proteins. Again unlike wild-type, this equilibrium mixture of EPR signals was not affected by increasing the electron flux, i.e., increasing the Fe protein:MoFe protein molar ratio to 5:1 (Table 1). Under either electron flux regime, there was always more residual signal 1a intensity and less signals 1b and 1c intensity for both variant MoFe proteins compared to wild-type. Neither signal 1b nor signal 1c were produced when the aQ191K MoFe protein was used and signal 1a accounted for all spectral intensity under both electron flux regimes (Table 1). No EPR signals, which could be attributed to P cluster oxidation [39,42], were observed with any of the MoFe proteins under low electron flux conditions. 3.2. Stopped-flow spectrophotometry – longer-time 430-nm absorbance changes Both the rate of primary inter-molecular electron transfer [20,38] and the absorbance changes that occur at 430 nm at longer times (ca. 200 ms after mixing for wild-

Fig. 2. Time course of both formation of the EPR signals 1b and 1c and loss of EPR signal 1a during turnover of the aH195Q MoFe protein in the presence of a 3-fold molar excess of wild-type Fe protein (signal 1a, red; signal 1b, black; and signal 1c, blue). Signal amplitude expressed as a percentage of the intensity of the g = 4.3 EPR signal of the aH195Q MoFe protein after mixing with buffer alone. The aH195Q MoFe protein was 60 lM after mixing. The lines are visual aids only.

type; [39]) were measured for all three variant and wild-type MoFe proteins under N2. All primary electron transfer rates (170 ± 15 s 1) were essentially identical. Initially, the increase in absorbance at 430 nm was studied as a function of increasing electron flux, using a range of Fe protein:MoFe protein molar ratios from 1:1 to 8:1, with wild-type MoFe protein. The 430-nm absorbance increase was observed only at fluxes generated by a 4:1 molar ratio or greater and it saturated at an 8:1 molar ratio (Fig. 3). At low flux under N2, no 430-nm absorbance increase was observed with the aH195Q, aH195N, and aQ191K MoFe proteins, however, at a 8:1 molar ratio, both the aH195Q and aH195N MoFe proteins showed the 430-nm absorbance increase, which started earlier (at ca. 100 ms) and ended sooner (by ca. 200 ms) than with wild-type MoFe protein (Fig. 4). The aQ191K MoFe protein did not exhibit this absorbance increase within 2 s of mixing under any conditions tried. 3.3. Freeze-quench Mo¨ssbauer spectroscopy of MoFe protein prosthetic groups

Fig. 1. Production of EPR signals 1b and 1c by the aH195Q MoFe protein during turnover under N2 with a 3-fold molar excess of wild-type Fe protein. The g = 4 components of signals 1a, 1b and 1c are indicated on the figure. Data collected at 22 ms (black), 102 ms (green), 180 ms (blue), and 350 ms (red), after mixing. The aH195Q MoFe protein was 60 lM after mixing.

Mo¨ssbauer spectra were collected on a 3:1 molar ratio mixture of wild-type Fe protein and 57Fe-labeled MoFe protein (84 lM after mixing) in the presence of 9 mM ATP, 10 mM Na2S2O4, 10 mM MgCl2 in 25 mM HEPES pH 7.4 at various times after initiating turnover under N2 at 23 °C. Fig. 5 shows the spectra after 10 ms (panels A– B), 36 ms (panels C–D), and 220 ms (panels E–F) after mixing. Spectrum A shows the 10-ms data plus the contribution (solid line) from the P clusters in their diamagnetic resting-state, sometimes referred to as PN. Subtraction of this P cluster contribution leaves spectrum B, which represents the contribution of the resting-state S = 3/2 FeMo-cofactors, sometimes referred to as MN. Similar subtraction from the 36-ms data (spectrum C) produces spectrum D and from the 220-ms data (spectrum E) produced

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Table 1 Equilibrium intensities of EPR spectral componentsa and comparative catalytic activities of wild-type, aH195Q, aH195N, and aQ191K MoFe proteins MoFe protein

Signal 1a

Signal 1b

Signal 1c

Reduce H+ and C2H2?

Bind N2?

Reduce N2?

Wild-type aH195Q aH195N aQ191K

4 23 38 100

79 64 52 0

17 13 10 0

Yes Yes Yes Yes

Yes Yesb Yesc No

Yes 1% of wtb Noc No

a b c

Achieved after 350 ms in the presence of a 5-fold molar excess of wild-type Fe protein and shown as a percentage of total EPR-signal intensity. See Ref. [43]. See Ref. [44].

Fig. 3. Stopped-flow spectrophotometry of the MgATP-induced electron transfer from wild-type Fe protein to wild-type MoFe protein under 100% N2 at the indicated protein molar ratios. Traces were obtained at 430 nm after a pre-equilibrated mixture of MoFe protein (20 lM) and Fe protein (20–160 lM) was mixed with MgATP in the presence of excess sodium dithionite.

tions of the resting-state (solid line) and reduced state (dashed line). Panel G, which was used to verify this analysis, shows a difference spectrum obtained from comparison of the 220-ms and 10-ms spectra. If the P cluster contribution to both spectra really is the same (and so cancels out on subtraction), the remaining data should only reflect the spectral difference between the reduced and resting-states of the FeMo-cofactor populations. Indeed, a simulation (solid line superimposed on spectrum G), which is representative of this difference, clearly describes spectrum G and so verifies our analysis. Fig. 6 shows the calculated distribution of Fe among the two prosthetic groups. At 10 ms, 53% resides in the restingstate P clusters and 47% in the resting-state FeMo-cofactors. At 36 ms after mixing, more than half of the FeMocofactor population has been reduced and, by 220 ms, the equilibrium situation has been achieved in which ca. 70% of the FeMo-cofactor population is reduced. Over this same time period, the P cluster population remained unchanged, showing neither oxidation nor reduction. 4. Discussion

Fig. 4. Stopped-flow spectrophotometry of the MgATP-induced electron transfer from wild-type Fe protein to various MoFe proteins under 100% N2 at higher electron flux. The MoFe proteins used: wild-type (black), aH195Q (blue), aH195N (green), and aQ191K (red). Traces were obtained at 430 nm when pre-equilibrated MoFe protein (20 lM) and Fe protein (160 lM) were mixed with MgATP in the presence of excess sodium dithionite.

Spectrum F. Both spectrum D and spectrum F represent a mixed population of resting-state and reduced FeMocofactors. The lines above spectra D and F show the deconvolution of these spectra into the individual contribu-

The spatial relationship of the two substituted aminoacid residues to the FeMo-cofactor is shown in Fig. 7. We have studied the effects of substitutions at these two positions on nitrogenase function in some detail previously [14,26,27,32–34,43,44]. This information led us to chose the aH195Q, aH195N and aQ191K MoFe proteins as suitable vehicles to assist in determining the potential function(s) of the three-electron-reduced FeMo-cofactor-based species responsible for the new S = 3/2 signal that we call signal 1b. If this species were functional, its production (and appearance of signal 1b) might correlate with some aspect of catalytic activity. Of the three, only the aH195Q and aH195N MoFe proteins developed signal 1b during turnover at low electron flux. This signal indicates that these two variants attain the three-electron-reduced redox level [31], which is required for N2 binding [20]. Thus, the appearance of signal 1b should correlate with an ability to bind N2 and it does (Table 1). During enzyme turnover, the aH195Q MoFe protein can bind and reduce N2 but only at ca. 1% of the wild-type rate [43]; the aH195N variant binds, but cannot reduce, N2 [44]; but the aQ191K variant is incapable of any interaction with N2 [44]. Further, the more rapid production of signal 1b with the aH195Q

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Fig. 5. Mo¨ssbauer spectra of rapid-freeze quenched samples from 57Fe-labelled wild-type MoFe protein during turnover under low electron flux. The data were recorded at 4.2 K in the presence of a 50-mT field applied parallel to the c-beam and collected after 10 ms (panels A–B), 36 ms (panels C–D), and 220 ms (panels E–F). The solid line plotted over spectra A, C and E corresponds to the theoretical spectrum of the resting-state P clusters. Spectra B, D and F result from subtraction of this resting-state P cluster contribution from spectra A, C and E, respectively. The solid line superimposed on spectra B, D and F is the sum of the theoretical spectra of the resting and reduced states of FeMo-cofactor and corresponds to the percentages presented in Fig. 6. Individual contributions of the resting (solid line) and reduced (dashed line) states of FeMo-cofactor are plotted over spectra D and F. Spectrum G is a difference spectrum (E A) that shows the conversion of resting-state (decreased in intensity and so upward-pointing absorption signals) into reduced state (increased in intensity and so downward-pointing quadrupole doublets) FeMo-cofactor and the superimposed solid line is the theoretical simulation representative of the difference between these two states.

and aH195N MoFe proteins, but in lower concentration, compared to wild-type suggests that the three-electron-

reduced MoFe protein species is accumulating in these variants because they are unable (or very slow) to go on

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Fig. 6. Time evolution of 57Fe Mo¨ssbauer absorptions from the FeMocofactor and the P cluster within the wild-type MoFe protein during turnover. A 3-fold molar excess of wild-type Fe protein under N2 was used. The absorptions are shown as a percentage of total absorption; resting-state FeMo-cofactor (black); reduced FeMo-cofactor (red); resting-state P cluster (blue).

Fig. 7. Environment around the FeMo-cofactor showing the location of the amino-acid residues (a-Gln191 and a-His195) substituted in this study. The component Mo (pink), Fe (green), and sulfur (yellow) atoms of FeMo-cofactor and the nitrogen (blue) atoms of the surrounding residues are highlighted.

to the four-electron-reduced species, where N2 would be irreversibly committed to NH3 production [20]. No EPR signals attributable to changes in the P clusters’ electronic status were visible in any of our low-electron flux spectra, even though electrons had obviously reached the FeMo-cofactor. Because crystal structures of 2:1 nitroge-

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nase complexes [5,6] show that the P cluster resides on a line between the Fe protein’s [4Fe–4S] cluster and the FeMo-cofactor, we decided to probe the question of whether the P clusters are an integral component of the electron transfer pathway from the Fe protein to the FeMo-cofactor. Previous stopped-flow spectrophotometric studies, supported by EPR data, suggested that an absorbance increase at 430 nm, which occurs ca. 200 ms after mixing, might arise from oxidation of the P clusters during turnover at higher electron flux under N2 [39]. We, therefore, conducted similar experiments on our variant nitrogenases. Neither wild-type (Fig. 3) nor any of the variants showed this longer-time 430-nm absorbance increase under low electron flux. Moreover, although the wild-type, aH195Q and aH195N MoFe proteins did show this 430-nm absorbance increase at higher flux under N2 (Fig. 4), the aQ191K variant did not. As an aside, the more rapid appearance of this 430-nm absorbance increase for the aH195Q and aH195N MoFe proteins is consistent with their more rapid production of EPR signal 1b. Based on the prior conclusions [39], these observations suggest that the P clusters may only oxidize when the more-reduced redox levels of the MoFe protein are achieved. If so, then the FeMo-cofactor is being reduced (because signal 1b is observed) at low electron flux but the P cluster may not oxidize. Is it then possible that electrons from the Fe protein are transferred directly to the FeMo-cofactor, by-passing the P clusters, at low electron flux? To gain insight into this question, we used rapid-freeze Mo¨ssbauer spectroscopy to collect data on a 3:1 molar ratio mixture of wild-type Fe protein and 57Fe-labeled wild-type MoFe protein over a 1-s period after initiating turnover under N2. At equilibrium, which was achieved by 220 ms, ca. 70% of the FeMo-cofactors present within the MoFe protein were reduced, but the population of P clusters in their resting-state was unchanged and remained so throughout the time course of our observations (Fig. 6). Because a change in the P cluster population due to redox activity would have been detected, this result supports the idea that, particularly at low flux, an alternative electron transfer pathway from the Fe protein to the FeMo-cofactor may exist and that this alternative pathway by-passes the P clusters. Finally, although crystal structures of the 2:1 nitrogenase complex [6–8] have provided the basis for the generally accepted electron transfer pathway through the P clusters, there are significant differences among them. One structure purports to show a ‘‘pre-encounter’’ complex of the Fe protein and MoFe protein [8] – how many other modes of interaction (as yet undiscovered) might there be (see, for example, [45])? The P clusters may have a N2 reduction-specific role in nitrogenase catalysis. Only at higher electron fluxes, conditions under which the most highly reduced redox levels of the MoFe protein are reached, do the P clusters oxidize and, when they do, it provides the extra ‘‘push’’ necessary to commit the initial reversibly bound N2 to irreversible reduction.

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