Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential

Elucidating how proteins control the reduction potentials (E-0') of [Fe-S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe-S] cluster E-0' (100-200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH-S bonds. The structures, combined with detailed investigation of the variation of E-0' with pH and temperature, show that the largest increases in E-0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50-100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+-Ndelta- dipole toward the reduced form of the cluster.