PubMed · 114520
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M J Chamberlin, W C Nierman, J Wiggs, N Neff. 1979-10-25. A quantitative assay for bacterial RNA polymerases.. https://pubmed.ncbi.nlm.nih.gov/114520/
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During the lag period before H2 is evolved by the nitrogenase system, the EPR signal of dinitrogenase decreases steadily, indicating transfer of electrons into dinitrogenase. The rate constant for the decrease in amplitude of the EPR signal, the steady state rate of H2 evolution from nitrogenase, and the length of the lag period have been measured. The data suggest that H2 is evolved only after dinitrogenase has been reduced by 2 electrons/molybdenum. The electrons that have been transfered into dinitrogenase during the lag period are not evolved as H2 upon denaturation of dinitrogenase. The existence of a lag indicates that the two nitrogenase proteins dissociate after every electron transfer. The lag occurs and the nitrogenase proteins dissociate under a variety of conditions of pH and temperature.
Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98, and 1.92 with t1/2 approximately 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1, and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0, and 1.98 is observed. The use of Azotobacter nitogenase components enriched selectively with 57Fe or 95Mo, as well as the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of high-potential iron-sulfur proteins and ferredoxins, respectively. The "no-CO" signal may be either an additional center or the N2-reducing site with H2 bound competitively.
In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe--2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO--Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of 'conformational protection' in this organism.