A serine->cysteine ligand mutation in the high potential iron-sulfur protein from Chromatium vinosum provides insight into the electronic structure of the [4Fe-4S] cluster

We have succeeded in preparing a mutant of the High Potential Iron-Sulfur Protein (HiPIP) from Chromatium vinosum in which a cysteine ligand has been replaced by a serine (C77S). Proton chemical shift data and nuclear Overhauser effects indicate that structural perturbations induced by the C77S mutation are minimal in both the oxidized and reduced forms of the HiPIP. The reduction potential of C77S is 25 mV lower than that of the wild-type HiPIP (WT) (0.2 M ionic strength, pH 4.5-9.0, 25 degrees C). Assignment of the hyperfine shifted signals in the H-1 NMR spectrum of oxidized C77S revealed that the temperature dependences of the signals associated with cluster-ligating residues 46 and 77 are Curie and and-Curie type, respectively, and are thus the reverse of those in WT. Taken together, these observations indicate that the iron bound to Ser-77 is less reducible than the corresponding iron in WT. The results are consistent with a previous model of the electronic structure of oxidized HiPIP clusters, confirming the presence of an equilibrium between two species of differing valence distributions. The current results permit the extension of this model to predict the relative reduction potentials of the individual iron ions in the oxidized HiPIPs up to now investigated.