In Azotobucter vinelandii MoFe protein the oxidation of the P clusters to the S = 7/2 state is associated with a redox reaction with Em,7 = +90 2 10 mV (vs the normal hydrogen electrode), n = 1. A concomitant redox process is observed for a rhombic S = 1/2 EPR signal with g = 1.97, 1.88 and 1.68. This indicates that both S = 1/2 and S = 7/2 signals are associated with oxidized P clusters occurring as a physical mixture of spin states. The maximal intensity of the S = 1/2 and S = 7/2 signals in the mediated equilibrium redox titration is similar if not identical to that of solidthionine-treated samples. Summation of the spin concentration of the S = 1/2 spin state (0.25 2 0.03 spin/ad2) and the S = 7/2 spin state (1.3 * 0.2 spida2p2) confirms that the MoFe protein has absolutely no more than two P clusters. In spectra of enzyme fixed at potentials around -100 mV a very low-intensity g = 12 EPR signal was discovered. In parallel-mode EPR the signal sharpened and increased >lO-fold in intensity which allowed us to assign the g = 12 signal to a non-Kramers system (presumably S = 3). In contrast with the non-Kramers EPR signals of various metalloproteins and inorganic compounds, the sharp absorption-shaped g = 12 signal is not significantly broadened into zero field, implying that the zero field splitting of the non-Kramers doublet is smaller than the X-band microwave quantum. The temperature dependence of this g = 12 EPR signal indicates that it is from an excited state within the integer spin multiplet. A bell-shaped titration curve with Em,7 = -307 * 30 mV and + 81 2 30 mV midpoint potentials is found for the g = 12 EPR signal. We propose that this signal represents an intermediate redox state of the P clusters between the diamagnetic, dithionite-reduced and the fully oxidized S = 7/2 and S = 1/2 state. Redox transitions of two electrons (-307?30mV) and one electron (+90?10mV) link the sequence S = O*S = 3+(S = 7/2 and S = U2). We propose to name the latter paramagnetic oxidation states of the P clusters in nitrogenase POx1 and POx2, and to retain PN for the diamagnetic native redox state. The magnetic circular dichroism and Mossbauer data on thionine-oxidized MoFe protein have to be re-evaluated bearing in mind that the oxidized P clusters can exist in two redox-states. Finally, an account is given of the EPR spectroscopic properties of S = 9/2 and other systems obtained upon superoxidation of the MoFe protein.Nitrogenase is the biological catalyst for the activation of the dinitrogen molecule in aqueous solution. The enzyme complex consists of two dissociable metalloproteins, the =230-kDa ad2 tetrameric MoFe protein and the homodimeric = 62-kDa Fe protein. Substrate binding, activation and reduction takes place on the MoFe protein, presumably

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni±Fe hydrogenase. Ten ORFs (mbhA±I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni±Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe±4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe±4S] protein. With P. furiosus soluble [4Fe±4S] ferredoxin as the electron donor the membranes produce H 2 , and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H 2 evolution to H 2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E m < 20.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni±Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H 2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H 2 ) connected by devices for transduction, transfer, recovery and safety-valving of energy.

No abstract

Thionine-oxidized nitrogenase MoFe proteins from Azotohucter vinelundii. Azotobacter cliroococcurn and Klehsiella pneurnoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin01 cm-' and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g z 10, z 2.5 and z 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters.Quantification relative to the S = 312 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, P, and Pb, only one of which is further oxidized with thionine resulting in the S = 7 / 2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithionite as the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant. Two models could describe the experimental data of the reduction by dithionite. The first model, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complex of 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociation of the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 400 s-1) was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.