Reversible Super-Reduction of the Cubane [4Fe-4S](3+;2+;1+) in the High-Potential Iron-Sulfur Protein Under Non-Denaturing Conditions. EPR Spectroscopic and Electrochemical Studies

Abstract

The reversible 2 x 1 e- reduction of the cubane cluster from oxidized to reduced to super-reduced states ([4Fe-4S]3+<-->[4Fe-4S]2+<-->[4Fe-4S]1+) was studied in high-potential iron-sulfur proteins (HiPIPs). Super-reduction to the 1+ state was not observed in any of the seven HiPIPs tested during cyclic voltammetry (down to -0.95 V). However, equilibration at low potential (pH 7.5) of Rhodopila globiformis HiPIP yields a transient peak around -0.47 V due to the oxidation of super-reduced HiPIP adsorbed at the electrode. The peak area depends on the equilibration potential according to a one-electron Nernst curve with a half-wave potential at -0.91 V. Reduction of R. globiformis HiPIP with titanium (III)citrate at pH 9.5 is very slow [pseudo-first-order half-life of 23 min with a 100-fold excess Ti(III)] but is reversible, and the EPR spectrum with g values of 2.04 and 1.92 is similar to that of reduced [4Fe-4S]1+ ferredoxins. Chemical or electrochemical reoxidation of the super-reduced form resulted in an EPR spectrum with g parallel = 2.12 and g perpendicular = 2.03, i.e. identical to that of oxidized HiPIP. From the equilibrium concentration of super-reduced HiPIP at a low concentration of Ti(III), a reduction potential of -0.64 V can be estimated. Super-reduction of the large HiPIP (iso-2) from Rhodospirillum salinarum is also possible with Ti(III)(gz = 2.05) but the super-reduced state is unstable. No super-reduction with Ti(III) was observed for the other HiPIPs. The difference between the electrochemically observed reduction potential and oxidation potential is explained by a fast and reversible conformational change upon super-reduction. The rate of super-reduction with Ti(III) is limited by the small amount (0.1%) of HiPIP in the 2+ state with the super-reduced conformation.