Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9446-9450, December 1988 Biochemistry Hydrogen burst associated with nitrogenase-catalyzed reactions (dinitrogenase/H2 evolution/molybdenum stoichiometry/substrates) JIHONG LIANG AND ROBERT H. BURRIS* Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706 Contributed by Robert H. Burris, September 12, 1988

ABSTRACT We have used a membrane-leak mass spec- inhibitor of N2 reduction by nitrogenase (9). This H2 inhibi- trometer to follow the time courses of H2 evolution and tion is specific for N2 reduction and has no effect on either the substrate reduction by nitrogenase [reduced ferredoxin:dini- reduction of substrates other than N2 or the reduction of trogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. In the protons. When the N2-reducing nitrogenase system is fur- absence of added substrates, dinitrogenase passes all of its nished with D2 (i.e., 2H2), HD is a product of the enzymatic electrons to protons to form H2, but when a reducible substrate reaction (10). In contrast to H2 evolution, which expends two is added the electrons from dinitrogenase are shared between electrons for one H2 produced, the formation of each HD protons and the added substrate so that the steady-state rate of molecule requires one electron, and the reaction absolutely H2 production is decreased. If a reducible substrate is added requires the presence of N2 (11, 12). The suggestion has been before the nitrogenase reaction is initiated, a pre-steady-state made that HD arises from decomposition of some partially burst of H2 is evident upon initiation ofthe reaction. This burst reduced intermediate in the N2 fixation process (10, 13). It is associated with all the substrates of nitrogenase examined- was reported (14) that dinitrogenase, in the absence of i.e., N2, N20, C2H2, NaN3, and NaCN. The H2 burst is dinitrogenase reductase, catalyzes the reduction of methyl- stoichiometric with dinitrogenase, but not with dinitrogenase ene blue and other oxidants with H2. The presence of H2 in reductase. In the H2 burst phase, 1 H2 is evolved per dinitro- the atmosphere above the dinitrogenase provided better genase molybdenum. Although a change in the ratio of nitro- protection against 02 inactivation. This observation indicates genase components changed the initial rate of the H2 burst, the that H2 can serve as an electron donor to nitrogenase under stoichiometry was not affected. Production of H2 by the burst appropriate (apparently nonphysiological) conditions. in the presence of a high concentration of substrate is termi- When the electron flux through dinitrogenase is low, and it nated after production of 1 H2 per dinitrogenase molybdenum, then is placed under an atmosphere of C2H2, there is a burst and a steady-state rate of H2 production is established. This of H2 production complementary to the lag phase before the response suggests that the H2 burst is not a catalytic event but linear rate of C2H4 production is established (15, 16). It has a result of a once-only activation process. been suggested that this H2 burst is a catalytic process, because its amplitude is much greater than predicted for a Nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase single enzyme turnover. The H2 burst also is observed at high (ATP-hydrolyzing), EC 1.18.6.1] is composed of two elec- electron flux through dinitrogenase with N2 as substrate. The tron-transferring proteins, dinitrogenase (MoFe protein) and burst phase is shorter than the turnover time ofdinitrogenase, dinitrogenase reductase (Fe protein). Reduced dinitrogenase and the burst is equivalent to one H2 evolved per molybde- reductase transfers a single electron at a time to dinitrogenase num (17). In this communication, we report that a pre- with the concomitant hydrolysis of MgATP. Dinitrogenase, steady-state burst of H2 production is observable not only with its multiple iron-sulfur centers and its "FeMoco" (FeMo with N2, but with all substrates of nitrogenase tested. This cofactor) prosthetic groups, serves as an electron sink capa- burst is stoichiometric with the molybdenum contained in the ble ofreducing all ofthe substrates ofnitrogenase. In addition dinitrogenase of the reaction system. to reducing its physiological substrate N2 to NH3, nitrogenase also is capable of reducing a variety of low molecular weight MATERIALS AND METHODS compounds such as N20, C2H2, N-, CN-, methyl isocya- nide, cyclopropene, and analogs of some ofthese compounds Azotobacter vinelandii OP was grown at 30'C in a 300-liter (1, 2). Dihydrogen is intimately involved in nitrogenase fermentor on the nitrogen-free medium described by Strand- catalysis (3). In the absence of other reducible substrates, berg and Wilson (18). The nitrogenase proteins were prepared nitrogenase passes electrons to protons in the aqueous from frozen cell paste and were purified to specific activities medium to produce molecular hydrogen in an ATP- of 1256 and 1826 nmol of C2H4 produced per min per mg of dependent H2 evolution reaction (4). This H2 evolution is protein for dinitrogenase and dinitrogenase reductase, re- inhibited by various nitrogenase substrates in ways that differ spectively. The proteins were homogeneous as indicated by both qualitatively and quantitatively. Whereas high concen- denaturing polyacrylamide gel electrophoresis. Molecular trations ofthe alternative substrates such as C2H2, CN-, and weights of 245,000 and 60,500 were accepted for dinitrogen- N20 (5-7) will completely suppress H2 evolution, the phys- ase and dinitrogenase reductase, respectively (19). Molyb- iological substrate of nitrogenase, N2, cannot abolish H2 denum was determined to be 1.47 mol per mol of dinitrogen- evolution even at 50 atmospheres (1 atm = 101.3 kPa) of N2 ase by the method of Clark and Axley (20). (8). Studies ofH2 evolution under various partial pressures of The experiments were conducted with a Varian MAT 250 N2 and N20 indicate that at a relatively high PN20, H2 isotope-ratio mass spectrometer equipped with a 1.62-ml evolution becomes N2-dependent (6). Therefore, H2 evolu- membrane-leak reaction chamber (21). The chamber was tion is an inherent part of the N2 reduction reaction. Dihy- closed with a standard taper plug with a narrow-bore central drogen is not only a product of nitrogenase but also an hole; when filled with solution, this provided a diffusion barrier to air from outside while permitting addition of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: atm, atmosphere(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9446 Downloaded by guest on September 28, 2021 Biochemistry: Liang and Burris Proc. Natl. Acad. Sci. USA 85 (1988) 9447 reagents with a syringe and long needle. The reaction solution was transferred to the chamber with a plastic syringe, and it filled the entire chamber plus the central hole of the plug. A magnetic stirring bar mixed the chamber contents rapidly. Dissolved gases in the reaction solution diffused across a 0.5-mil (12.7-gm) Teflon membrane supported by a porous frit, through an evacuated stainless steel tube immersed in a dry-ice trap, and into the mass spectrometer. The mass spectrometer was set to monitor signals of mle 2 (H2) and 28 (N2 or C2H4) simultaneously and continuously. The reaction mixture contained 5.9 mM ATP, 15.4 mM magnesium acetate, 28 mM creatine phosphate, 0.1 mg of creatine kinase per ml, 20 mM Na2S204, 20 mM Tris and 50 mM Hepes (adjusted to pH 7.5), and nitrogenase proteins as indicated. Anaerobically prepared reaction mixture (without was with a to E ATP) transferred syringe vaccine-stoppered c bottles that contained gaseous substrate or Ar, and the bottles -6 0) were shaken at room temperature for 20-30 min to allow the 0 m equilibration ofthe gaseous substrates between liquid and gas 'a phases. Solutions of solid substrates, such as NaN3 and 0 NaCN, were added directly to the reaction mixture before it I- was transferred to the reaction chamber. The membrane-leak reaction chamber was made anaerobic by consecutive wash- ings with 20 mM Tris buffer/2 mM dithionite and flushing with Ar. The 1.8 ml of reaction solution transferred to the chamber filled the entire chamber plus the hole of the plug. The reactions were initiated by addition of MgATP. The mass spectrometer was calibrated for H2, N2, and C2H4 by injecting buffer containing known quantities of a dissolved gas into the solution in the chamber (usually Ar-flushed buffer). The peak height recorded by the mass spectrometer was proportional to the amount of dissolved gas added. A plot of moles of gas injected vs. peak heights had a correlation coefficient of 0.9999. Since the sample inlet line of the mass spectrometer is under vacuum, a part of the dissolved gases are continuously removed from the membrane-leak chamber 60 and consumed in the analysis. This systematic error is cor- Time, sec rected by an empirically derived diffusion curve applied with a personal computer. The relationship between the diffusion FIG. 1. Inhibition of nitrogenase-mediated H2 evolution by var- rate and the concentration of dissolved gas was established by ious substrates. Experiments were conducted with a membrane-leak injecting known quantities of dissolved gas and measuring the mass spectrometer. (A) Under N2; component ratio (molar ratio of initial slopes of the diffusion curves. dinitrogenase reductase to dinitrogenase), 0.76. Curves: a, under Ar; The response times of the membrane-leak mass spectrom- b, 0.01 atm of N2. (B) Under N20; component ratio same as in A. eter Curves: d, under Ar; e, 0.1 atm of N20; f, 1.0 atm of N20. (C) Under to H2, N2, and C2H4 were determined to be 0.46, 1.8, and C2H2; component ratio, 2.33. Curves: g, under Ar; h, 0.01 atm of 3.3 sec, respectively, by injection of dissolved gases into the C2H2; i, 0.04 atm of C2H2; j, 0.08 atm of C2H2 (D) With NaN3; reaction chamber. component ratio, 1.55. Curves: k, 0.47 mM NaN3; 1, 4.7 mM NaN3; Nitrogenase-mediated H2 evolution also was measured m, 18.5 mM NaN3; n, 182 mM NaN3. with a Clark-type electrode. Half-saturated KCI solution served as the electrolyte bathing the electrode, and the and B; this depended on the electron flux through dinitrogen- electrode was separated from the water-jacketed glass reac- ase, which was altered by changing the ratio of nitrogenase tion chamber by a 0.5-mil Teflon membrane (22). components. If we assume that the steady-state rate of H2 evolution is the rate of electron flux through the enzyme and RESULTS that this rate remains constant from the moment that the reaction is initiated, calculation reveals that the lag phases Time courses for H2 evolution in the presence or in the are equivalent to 9.6 and 8.1 electrons per molybdenum, absence of reducible substrates are presented in Fig. 1. Data respectively. This means that in the absence of substrate, H2 in the different subfigures were collected in different exper- is not released until the enzyme has accumulated at least 8 iments, whereas traces shown in each subfigure were re- electrons. H2 evolution was inhibited by all substrates tested corded in the same experiment under identical experimental in the experiments represented by Fig. 1. N2 is unable to conditions. In all cases, a linear rate of H2 evolution was completely block H2 evolution even under infinitely high achieved only after a lag or a burst phase. An extrapolation pressure (8). In 0.67 mM dissolved N2 (dissolved N2 under of the linear section of the reaction progress curve to the 1-atm N2 gas phase), H2 evolution still accounts for 33% of abscissa allows the measurement of the lag periods, whereas the total electron flux, ifwe assume that the total electron flux the H2 burst phase is considered to be from the base line to is not affected by N2. N20 att 21.2 mM (dissolved N20 under the point at which the extrapolations of steady-state rate and 1-atm N20 gas phase) substantially inhibited H2 evolution; the initial burst rate cross each other (e.g., see Fig. 3A). In the the electron flux for H2 production under such conditions (6.9 absence ofreducible substrate (reaction mixture sparged with electron pairs per mg per min) was 4.6% of the flux under Ar Ar), a lag phase always was observed before the steady-state (149.3 electron pairs per mg per min). C2H2 and NaCN (see rate of H2 production was established. The length of the lag Fig. 3B) are stronger inhibitors than the other nitrogenase phases varied from 5.2 sec in Fig. 1C to 10.7 sec in Fig. 1 A substrates. C2H2 at 3.5 mM (dissolved C2H2 under 0.08-atm Downloaded by guest on September 28, 2021 9448 Biochemistry: Liang and Bums Proc. Nati. Acad. Sci. USA 85 (1988)

PC2H2) reduced H2 evolution from 363.4 electron pairs per mg per min under Ar to 55 electron pairs per mg per min; 20 mM NaCN completely abolished H2 evolution after the initial H2 burst. Increase in NaN3 concentration decreased the electron allocation to proton reduction. Fig. 1 also shows that if the rate of H2 evolution was sufficiently suppressed, a burst phase, rather than a prolonged lag period, preceded the steady-state rate of H2 production with 21.2 mM N20 and 182 50, mM NaN3. However, a H2 burst was not observed with 0.67 mM N2. It appeared that there was no H2 burst under these 60 o conditions either because the.high H2 evolution rate masked a)B 0 the burst phase or because'the burst was specifically asso- B ciated with N20 and NaN3. To clarify the issue the H2 burst was examined with a variety of substrates (Figs. 2 and 3). A

H2 burst always was evident with all substrates, including N2, 30- N20, C2H2, NaN3, and NaCN. In all cases except NaCN, the amount of H2 produced in the burst period was very close to 1 H2 evolved per molybdenum in the reaction system (Table 1). The H2 burst in 20 mM NaCN apparently was >1 H2 per molybdenum. By proper setting ofthe mass spectrometer, we 0 were able to measure the concomitant production of N2 from 0 1 5 30 45 60 N20 or NaN3, or C2H4 from C2H2. In all experiments shown Time, sec in Fig. 2, the total gas production was linear after a short time lag. Although NaN3 yields products other than N2, such as FIG. 3. H2 burst with N2 or NaCN as substrate. (A) Under 2.0 atm NH3 and N2H4 (23-26), the distribution of electrons among of N2. (B) With 20 mM NaCN. The end of the burst phase (i.e., the these products presumably does not change dramatically point at which the extrapolations of steady-state rate and initial burst during the first minute of measurement of reduction of NaN3. rate intersect) is indicated in A. Thus, a constant rate of(N2 + H2) production should indicate least two electrons have to be transferred from dinitrogenase enzyme. The a constant rate of electron flow through the reductase to dinitrogenase before the release of any product linearity of the total activity suggests that the H2 burst is not (27). The electron flow between the components of nitrogen- due to an alteration in the enzyme activity. Note that there ase has been considered a rate-limiting process (28, 29), as always is a time lag before any product release as indicated dinitrogenase reductase dissociates from dinitrogenase after by extrapolation of the total activity curve to the x axis. At each single electron transfer and this dissociation is the 150 slowest step in the catalysis cycle. Therefore, a time lag before product release is logically expected, and the lower the electron flux through the enzyme, the longer the expected

100 time lag. Fig. 2 also shows that the lag periods before the establishment of constant rates ofN2 production from N20 or NaN3, or C2H4 production from C2H2, are much longer than more 50 those for the total activity. This suggests that highly reduced dinitrogenase is responsible for substrate reduction than for H2 production in the burst phase, and that H2 is the 120 first product to be released in nitrogenase-catalyzed reac- tions. The same type of experiments shown in Figs. 2 and 3 also were done with a Clark-type electrode to measure H2. A burst of H2 occurred before the steady-state rate of H2 evolution was established; 1.19 H2 per molybdenum were produced in the burst in 91 mM NaN3. The H2 burst is ATP-dependent and requires electron transfer between the in the 00 component proteins; no H2 production is detectable absence of ATP or either component protein of nitrogenase (data not shown). 90 90 9 /h We measured the amount of H2 produced during the burst C phase with respect to the component proteins of nitrogenase (Table 2). Either the amount of dinitrogenase was varied 60- while the amount of dinitrogenase reductase was kept con- stant, or the component ratio was kept constant but the

30 Table 1. H2 bursts with high concentrations of substrates Fe/MoFe Mo, H2 burst, Substrate component ratio nmol nmol H2/Mo 0 0.98 0 15 30 45 60 N20 (1 atm) 0.76 14.6 14.3 N2 (2 atm) 1.23 12.6 14.0 1.11 Time, sec C2H2 (2 atm) 1.55 10.8 9.1 0.85 NaN3 (91 mM) 1.59 12.0 14.3 1.19 FIG. 2. H2 burst with various substrates. (A) Under 1.0 atm of NaCN (20 mM) 1.23 12.3 28.2 2.29 N20. Curves; a, N2 plus H2; b, N2; c, H2. (B) With 314 mM NaN3. Curves: d, N2 plus H2; e, N2; f, H2. (C) Under 2 atm of C2H2. Curves: Experiments were performed with a membrane-leak mass spec- g, C2H4 plus H2; h, C2H4; i, H2- trometer. Downloaded by guest on September 28, 2021 Biochemistry: Liang and Burris Proc. Natl. Acad. Sci. USA 85 (1988) 9449 Table 2. Correlation between H2 burst and the concentration of dinitrogenase MoFe Fe H2 prot., prot., burst, H2/Fe Exp. nmol nmol Fe/MoFe nmol H2/Mo prot. 1 3.62 5.00 1.38 3.9 0.74 0.78 7.23 10.00 1.38 6.9 0.66 0.69 10.85 15.00 1.38 11.1 0.70 0.74 E 14.47 20.00 1.38 14.0 0.66 0.70 7.23 20.00 2.77 10.0 0.94 0.50 CL 5.42 20.00 3.69 6.0 0.76 0.30 cm) 3.62 20.00 5.52 5.6 1.04 0.28 2 5.85 3.75 0.64 9.9 1.16 2.64 20- 5.82 7.50 1.29 6.6 0.77 0.88 I~~~~~~~~~~~~~~~~~~~~ 5.77 15.00 2.60 7.2 0.85 0.48 5.70 25.00 4.39 7.6 0.92 0.30 Average 0.84 ± 0.16 10 All experiments were run under 1 atm of N20 with the membrane- leak mass spectrometer. MoFe prot., dinitrogenase; Fe prot., dini- trogenase reductase. protein concentrations were increased concomitantly (Fig. Time, sec 4). The amount of H2 produced in the burst ranged from 0.66 to 1.16 H2 per molybdenum and averaged 0.84 ± 0.16 H2 per FIG. 4. Relationship ofH2 burst to concentration ofdinitrogenase molybdenum. Therefore, the H2 burst appears stoichiometric and component ratio. Experiments were conducted under 1 atm of to the concentration ofthe active site. However, the H2 burst N20. The lag times have been deleted from the curves. (A) Compo- shows no correlation with the concentration of nent ratio was constant at 1.38, but total protein concentrations dinitrogenase varied. The quantities (in nmol) of dinitrogenase in each reaction reductase; the amount of H2 produced in the burst per mixture were 3.62, 7.23, 10.85, and 14.47 for curves a-d, respec- molecule of dinitrogenase reductase varied from 0.28 to 2.64. tively. (B) Dinitrogenase reductase was kept constant at 20 nmol in Changing the electron flux through the enzyme by altering the each reaction mixture, but dinitrogenase varied: 14.47, 7.23, and 3.62 component ratio had no effect on the stoichiometry of the H2 nmol for curves d-f, respectively. burst, although it did change the total enzymatic activity. tions ofN2 and C2H2 demonstrated a H2 burst unambiguously (Figs. 2 and 3). Apparently a H2 burst always occurs after DISCUSSION each initiation of the enzymatic reaction, regardless of the When provided energy and reducing power, nitrogenase nature and the concentration ofthe substrate, and even under keeps turning over by converting protons in the medium to Ar. However, because the initial rate of the H2 burst may be H2. This H2 evolution reaction is inhibited by all physiolog- relatively low, the H2 burst may not be recognizable until the ical (N2) and nonphysiological (N20, C2H2, NaN3, NaCN) steady-state rate of H2 evolution is suppressed below the substrates tested; some of them, at high concentrations, initial rate of the H2 burst. We have examined most of the completely eliminate H2 production and divert all electrons to known substrates of nitrogenase for their support of a H2 their own reduction. The rapid and continuous measurements burst. All substrates examined support a H2 burst. With 2 atm of time courses of substrate reduction and ofconcomitant H2 of C2H2 and 20 mM NaCN, after the initial H2 burst there is evolution presented in this communication provide detailed no further production of H2. Parallel experiments were information about the pre-steady-state and steady-state ki- conducted with the membrane-leak mass spectrometer and netics of H2 evolution and substrate reduction and about the with a Clark-type electrode. Each technique gave essentially interactions between H2 evolution and substrate reduction. the same results. This furnishes assurance that the observed In the absence of any added substrate, all electrons flowing H2 burst is indeed a property of nitrogenase-catalyzed reac- through nitrogenase are evolved as H2. A time lag, whose tions, not an artifact. length varies depending on the electron flux through the That -1 H2 is evolved per molybdenum, and that there is enzyme, always precedes the steady-state rate of H2 produc- no correlation between the amplitude of the H2 burst and the tion. This time lag, examined under standardized conditions, concentration of dinitrogenase reductase, suggests that dini- is longer in the absence of than in the presence of reducible trogenase is the site of H2 production. The component ratio substrate. If the time lag represents the accumulation of (16) and concentration (28, 29) of nitrogenase play an impor- reducing equivalents in dinitrogenase before product release tant role in controlling the electron allocation among prod- (27), one could suggest (i) that at least two forms of dinitro- ucts. One might argue that the stoichiometry of 1 H2 evolved genase are capable of evolving H2 and (ii) that reducible in the H2 burst per molybdenum could arise from a fortuitous substrates of nitrogenase can influence the distribution of choice of a protein concentration and a component ratio. these different forms in the total population ofdinitrogenase. However, we have performed experiments with increasing In the presence of substrate, the dominating form(s) of protein concentrations at a fixed component ratio, and with H2-evolving dinitrogenase requires fewer electrons before H2 changing component ratios and a constant nitrogenase con- is released than does the form(s) in the absence of substrate. centration. The results indicate that although a change in the Interestingly, when the potential rate of H2 evolution is component ratio of nitrogenase proteins altered the total suppressed to low levels by reducible substrate, a burst of H2 enzymatic activity and the initial rate of the H2 burst, the is observed upon initiation of nitrogenase action and before stoichiometry of 1 H2 per molybdenum in the burst remained the steady-state rate is established. The failure to induce a H2 relatively constant and within the experimental error of the burst under 1 atm of N2 or 0.08 atm of C2H2 (Fig. 1) methods. Simpson (17) observed that there was a H2 burst in apparently is due to the low concentration of substrate used. 50 ,uM N2 (about 0.08 atm of N2), in which 1 H2 was evolved Repeating the experiments of Fig. 1 with higher concentra- per molybdenum. Thorneley and Eady (15) observed a burst Downloaded by guest on September 28, 2021 9450 Biochemistry: Liang and Burris Proc. Natl. Acad. Sci. USA 85 (1988) phase of H2 evolution complementary to the lag phase for 2. Burgess, B. K. (1985) in Molybdenum Enzymes, ed. Spiro, C2H4 production under C2H2 at 10TC. Since its magnitude was T. G. (Wiley, New York), pp. 161-219. much greater than could be attributed to a single enzyme 3. Simpson, F. B., (1987) Physiol. Plant. 69, 187-190. turnover, they concluded that it represented a catalytic 4. Bulen, W. A., Burns, R. C. & LeComte, J. R. (1965) Proc. Natl. Acad. Sci. USA 53, 532-539. process. However, their "burst" is not the same as the burst 5. Rivera-Ortiz, J. M. & Burris, R. H. (1975) J. Bacteriol. 123, we report here. Their "burst" spanned about 5 min; in 537-545. contrast, the burst described in this paper was completed in 6. Liang, J.-H. & Burris, R. H. (1988) Biochemistry 27, 6726- 2-10 sec. Lowe and Thorneley (28) observed a H2 burst under 6732. N2 with a rapid quenching technique. The conditions used in 7. Jensen, B. B. & Burris, R. H. (1986) Biochemistry 25, 1083- their experiment were very similar to those ofour experiment 1088. 1, line 7 of Table 2; the ratio of dinitrogenase reductase to 8. Simpson, F. B. & Burris, R. H. (1984) Science 224, 1095-1097. dinitrogenase was 4:1 and the temperature was 23TC. Calcu- 9. Wilson, P. W. & Umbreit, W. W. (1937) Arch. Mikrobiol. 8, lation from their data indicates that 1.1 H2 was evolved per 440-457. molybdenum during the burst phase. They speculated that in 10. Hoch, G. E., Schneider, K. C. & Burris, R. H. (1960) Biochim. Biophys. Acta 37, 273-279. the burst period, H2 is released by a form of the enzyme, 11. Guth, J. H. & Burris, R. H. (1983) Biochemistry 22, 5111-5122. E2H2. They suggested that after the burst phase, the nitro- 12. Li, J.-L. & Burris, R. H. (1983) Biochemistry 22, 4472-4480. genase occurs as several enzyme species they designated as 13. Bulen, W. A. (1976) in Proceedings of the 1st International E3, E4, E5, E6, and E7, with each form binding N2 or a Symposium on Nitrogen Fixation, eds. Newton, W. E. & reduction intermediate of N2. With formation of these inter- Nyman, C. J. (Washington State Univ. Press, Pullman, WA), mediates, the concentration of E2H2 and the rate of H2 pp. 177-186. evolution decreased. They apparently did not recognize that 14. Wang, Z.-C. & Watt, G. D. (1984) Proc. Natl. Acad. Sci. USA 1 H2 is produced per molybdenum in the burst period. If H2 81, 376-379. is indeed produced by E2H2, one must ask, why is 1 H2 15. Thorneley, R. N. F. & Eady, R. R. (1977) Biochem. J. 167, produced per molybdenum, not more or less than 1? Chatt 457-461. (30) proposed that the molybdenum of dinitrogenase is 16. Hageman, R. V. & Burris, R. H. (1980) Biochim. Biophys. Acta the backbone. He 591, 63-75. situated in the pocket provided by protein 17. Simpson, F. B. (1985) Ph.D. Thesis (Univ. of Wisconsin- suggested that as soon as the electron flow is established Madison). between the nitrogenase components, the first reaction is to 18. Strandberg, G. W. & Wilson, P. W. (1968) Can. J. Microbiol. scavenge protons from the walls of the pocket until the rate 14, 25-30. of proton diffusion into the pocket is slow enough to allow 19. Swisher, R. H., Landt, M. & Reithel, F. J. (1975) Biochem. reduction of the molybdenum site to the state necessary for Biophys. Res. Commun. 66, 1476-1482. substrate binding. Unless fortuitously the free protons in the 20. Clark, L. J. & Axley, J. H. (1955) Anal. Chem. 27, 2000-2003. pocket are 2 per molybdenum before the initiation of the 21. Hoch, G. & Kok, B. (1963) Arch. Biochem. 101, 160-170. reaction, and no additional protons enter thereafter, this 22. Sweet, W. J., Houchins, J. P., Rosen, P. R. & Arp, D. J. (1980) the the Anal. Biochem. 107, 337-340. hypothesis likewise cannot explain stoichiometry of 23. Schollhorn, R. & Burris, R. H. (1967) Proc. Natl. Acad. Sci. H2 burst. Probably the most plausible explanation is that a USA 57, 1317-1323. once-only activation step rather than sustained catalytic 24. Hardy, R. W. F. & Knight, E., Jr. (1967) Biochim. Biophys. activity is responsible for the H2 burst (31, 32). The H2 burst Acta 139, 69-90. is only a manifestation of such an activation process. Once 25. Dilworth, M. J. & Thorneley, R. N. F. (1981) Biochem. J. 193, the enzyme is activated, it remains in this state and continues 971-983. to turn over as a normal enzyme. In the presence of high 26. Rubinson, J. F., Burgess, B. K., Corbin, J. L. & Dilworth, concentrations of C2H2 or NaCN, the H2 evolution is com- M. J. (1985) Biochemistry 24, 273-283. pletely abolished after the initial H2 production by the 27. Hageman, R. V. & Burris, R. H. (1979) J. Biol. Chem. 254, state of the could 11189-11192. activation process. The activated enzyme 28. Lowe, D. J. & Thorneley, R. N. F. (1984) Biochem. J. 224, be maintained by electron flow through the enzyme. When 877-886. dinitrogenase is drained of electrons, it may return to the 29. Thorneley, R. N. F. & Lowe, D. J. (1985) in Molybdenum ground state that characterizes the H2 burst. Enzymes, ed. Spiro, T. G. (Wiley, New York), pp. 221-284. P. & We appreciate the discussions and suggestions of Frank B. 30. Chatt, J. (1980) in Nitrogen Fixation, eds. Steward, W. D. Simpson concerning this research project. This work was supported Gallon, J. R. (Academic, London), pp. 1-18. by the College of Agricultural and Life Sciences, University of 31. Smith, B. E., Thorneley, R. N. F., Eady, R. R. & Mortenson, of Grant DE- L. E. (1976) Biochem. J. 157, 439-447. Wisconsin-Madison, and by Department Energy 32. Smith, B. E., Eady, R. R., Thorneley, R. N. F., Yates, M. G. FG02-87ER13707. & Postgate, J. R. (1977) in Recent Developments in Nitrogen 1. Postgate, J. R. (1982) The Fundamentals ofNitrogen Fixation Fixation, eds. Newton, W., Postgate, J. R. & Rodriguez- (Cambridge Univ. Press, Cambridge, UK). Barrueco, C. (Academic, London), pp. 191-203. Downloaded by guest on September 28, 2021