Hydrogen burst associated with nitrogenase-catalyzed reactions.

Abstract

We have used a membrane-leak mass spectrometer to follow the time courses of H2 evolution and substrate reduction by nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. In the absence of added substrates, dinitrogenase passes all of its electrons to protons to form H2, but when a reducible substrate is added the electrons from dinitrogenase are shared between protons and the added substrate so that the steady-state rate of H2 production is decreased. If a reducible substrate is added before the nitrogenase reaction is initiated, a pre-steady-state burst of H2 is evident upon initiation of the reaction. This burst is associated with all the substrates of nitrogenase examinedi.e., N2, N20, C2H2, NaN3, and NaCN. The H2 burst is stoichiometric with dinitrogenase, but not with dinitrogenase reductase. In the H2 burst phase, 1 H2 is evolved per dinitrogenase molybdenum. Although a change in the ratio of nitrogenase components changed the initial rate of the H2 burst, the stoichiometry was not affected. Production of H2 by the burst in the presence of a high concentration of substrate is terminated after production of 1 H2 per dinitrogenase molybdenum, and a steady-state rate of H2 production is established. This response suggests that the H2 burst is not a catalytic event but a result of a once-only activation process.Nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is composed of two electron-transferring proteins, dinitrogenase (MoFe protein) and dinitrogenase reductase (Fe protein). Reduced dinitrogenase reductase transfers a single electron at a time to dinitrogenase with the concomitant hydrolysis of MgATP. Dinitrogenase, with its multiple iron-sulfur centers and its "FeMoco" (FeMo cofactor) prosthetic groups, serves as an electron sink capable of reducing all ofthe substrates ofnitrogenase. In addition to reducing its physiological substrate N2 to NH3, nitrogenase also is capable of reducing a variety of low molecular weight compounds such as N20, C2H2, N-, CN-, methyl isocyanide, cyclopropene, and analogs of some ofthese compounds (1,2). Dihydrogen is intimately involved in nitrogenase catalysis (3). In the absence of other reducible substrates, nitrogenase passes electrons to protons in the aqueous medium to produce molecular hydrogen in an ATPdependent H2 evolution reaction (4). This H2 evolution is inhibited by various nitrogenase substrates in ways that differ both qualitatively and quantitatively. Whereas high concentrations of the alternative substrates such as C2H2, CN-, and N20 (5-7) will completely suppress H2 evolution, the physiological substrate of nitrogenase, N2, cannot abolish H2 evolution even at 50 atmospheres (1 atm = 101.3 kPa) of N2 (8). Studies of H2 evolution under various partial pressures of N2 and N20 indicate that at a relatively high PN20, H2 evolution becomes N2-dependent (6). Therefore, H2 evolution is an inherent part of the N2 reduction reaction. Dihydrogen is not only a product of nitrogenase ...

Mechanism of Mo-Dependent Nitrogenase

“…For example, it is known that three or four electrons must accumulate within the MoFe protein before N 2 binds (E 3 or E 4 states) (33). Further, when N 2 binds to the MoFe protein, a stoichiometric quantity of H 2 is evolved (69, 70). In the absence of N 2, the MoFe protein under turnover cycles through low E n states while producing H 2 .…”

Nitrogen Fixation by Nitrogenases: A Quantum Chemical Study

“…With some substrates there is an initial burst of about one H 2 molecule per Mo, which could be due to the substrate displacing H 2 on binding. 17 The crystallographic work of the Rees and Bolin groups 13,14,18 has made the present study possible by providing a structural basis for discussing the N 2 -ase mechanism. In particular it has revealed the very surprising structure of FeMoco shown in Figure 1.…”

Investigation of the Photoheterotrophic Hydrogen Production of <i>Rhodobacter sphaeroides</i> KCTC 1434 using Volatile Fatty Acids under Argon and Nitrogen Headspace

“…The radical increase in pH could be explained by the nitrogenase-mediated H 2 production via the reaction (Liang and Burris, 1988;Kars and Gunduz, 2010 …”