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 (etp(Fd)) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemical methods, and Mossbauer, UV-Vis and circular dichroism spectroscopies. The Mossbauer spectrum is consistent with a standard diferric [2Fe-2S](2+) cluster. While showing sequence homology to vertebrate ferredoxins, the E degrees' and the reduction thermodynamics for etp(Fd) (-0.392 V) are similar to plant-type ferredoxins. Relatively stable Cys to Set derivatives were made for each of the four bound Cys residues and variations in the visible spectrum in the 380-450 nm range were observed that are characteristic of oxygen ligated clusters, including members of the [2Fe-2S] cluster IscU/ISU scaffold proteins. Circular dichroism spectra were similar and consistent with no significant structural change accompanying these mutations. All derivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to the reductase was similar, however, V-max reflecting rate limiting electron transfer was found to decrease similar to 13-fold. The data are consistent with relatively minor perturbations of both the electronic properties of the cluster following substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein. (C) 2011 Elsevier Inc. All rights reserved.