Phenylalanine 48 of Chromatium vinosum high potential iron protein is essential for stability of the oxidized [Fe 4S 4] cluster. Site-directed mutagenesis and NMR studies as a probe of cluster chemistry

Abstract

Point mutations of the conserved aromatic residue phenylalanide 48 (Phe48Arg.His) in Chromatium vinosum high potential iron protein have been examined with the aim of understanding the functional role of this residue. For both mutants the change in midpoint potential of the [Fe 4S 4] 3+/2+ cluster is minimal (< + 20 mV). The oxidized state is unstable relative to the recombinant native, with the observation of an ‘autoreduction’ pathway that appears to be mediated by degradation of the cluster in a fraction of the oxidized sample. This provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction results from the increased solvent accessibility of the cluster core in the non-conservative Phe48 mutants. Increased solvent access has been characterized by 1H 15N HMQC experiments.