Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein

Abstract

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

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

“…8, upper spectrum). From the position of the g values, this signal is strongly reminiscent of the signal interpreted by Tittsworth and Hales [40] as S = 1/2 signal arising from oneelectron-oxidized P-clusters (AvIMo protein). This signal was, however, absent in the case of the FeFe protein.…”

P-cluster maturation on nitrogenase MoFe protein

“…4B), dithionite-reduced Av1 ⌬nifZ⌬nifB(varied time) proteins show a decrease in the magnitude of the S ϭ 1 ⁄2 signal that has been previously assigned to the P-cluster precursor in the ''second half'' of the Av1 ⌬nifZ⌬nifB protein (19), whereas, when subjected to parallel-mode EPR analysis (Fig. 4C), indigo disulfonateoxidized Av1 ⌬nifZ⌬nifB(varied time) proteins display an increase in the intensity of the unique signal that is specific for the P-cluster in the P 2ϩ oxidation state (22,23), indicating an increase of mature P-cluster content as a result of the conversion of P-cluster precursor into fully assembled cluster. Over a time period of 60 min, the percentages of changes in perpendicular and parallelmode EPR signals ¶ are 32% and 41%, respectively, which correlate well with the 37% increase in activity (Fig.…”

Electrochemical study of the redox properties of [2Fe‐2S] ferredoxins Evidence for superreduction of the Rieske [2Fe‐2S] cluster

“…The third thesis is based on the two different transitions, 3 +/2 + and 2 +/1 +, observed in [4Fe-4S] proteins (i.e. high-potential iron proteins vs. ferredoxins), and on the multiple transitions observed in the putative [6Fe-6S] protein [33] and in the P-cluster of nitrogenase [34].…”