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 E_m,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 E_m,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 P^OX1 and P^OX2, and to retain P^N 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.