Redox Chemistry of the Schizosaccharomyces pombe Ferredoxin Electron-Transfer Domain and Influence of Cys to Ser Substitutions

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain(etpFd) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemicalmethods, and Mössbauer, UV–Vis and circular dichroism spectroscopies. The Mössbauer spectrum isconsistent with a standard diferric [2Fe–2S]2+ cluster. While showing sequence homology to vertebrateferredoxins, the E°' and the reduction thermodynamics for etpFd (−0.392 V) are similar to plant-typeferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues andvariations in the visible spectrum in the 380–450 nm range were observed that are characteristic of oxygenligated clusters, including members of the [2Fe–2S] cluster IscU/ISU scaffold proteins. Circular dichroismspectra were similar and consistent with no significant structural change accompanying these mutations. Allderivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to thereductase was similar, however, Vmax reflecting rate limiting electron transfer was found to decrease ~13-fold.The data are consistent with relatively minor perturbations of both the electronic properties of the clusterfollowing substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.