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

1993; Wiley; Volume: 212; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1993.tb17632.x

1432-1033

Antonio J. Pierik, Hans Wassink, H. Haaker, Wilfred R. Hagen,

Metalloenzymes and iron-sulfur proteins

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/α 2 β 2 ) and the S = 7/2 spin state (1.3 ± 0.2 spin/α 2 β 2 ) 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.