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

Abstract

In Azotobacter vinelandii MoFe protein the oxidation of the P clusters to the S= 7/2 state is associated with a redox reaction with Em,7.5=+90 ± 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 solid‐thionine‐treated samples. Summation of the spin concentration of the S= 1/2 spin state (0.25 ± 0.03 spin/α2β2) and the S= 7/2 spin state (1.3 ± 0.2 spin/α2β2) 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 > 10‐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,7.5=−307 ± 30 mV and +81 ± 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 ± 30 mV) and one electron (+90 ± 10 mV) link the sequence S= 0 ⇌S= 3 ⇌ (S = 7/2 and S= 1/2). 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 Mössbauer 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. Copyright © 1993, Wiley Blackwell. All rights reserved