PHENYLALANINE-48 OF CHROMATIUM-VINOSUM HIGH-POTENTIAL IRON PROTEIN ISESSENTIAL FOR STABILITY OF THE OXIDIZED [FE4S4] CLUSTER - SITE-DIRECTED MUTAGENESIS AND NMR-STUDIES AS A PROBE OF CLUSTER CHEMISTRY

Point mutations of the conserved aromatic residue phenylalanine 48 (Ph e48Arg,His) in Chromatium vinosum high potential iron protein have bee n examined with the aim of understanding the functional role of this r esidue. For both mutants the change in midpoint potential of the [Fe4S 4](3+/2+) cluster is minimal ( < +20 mV). The oxidized state is unstab le relative to the recombinant native, with the observation of an 'aut oreduction' pathway that appears to be mediated by degradation of the cluster in a fraction of the oxidized sample. This provides the reduci ng equivalents required to bring about reduction of the remainder of t he sample. This degradative reaction results from the increased solven t accessibility of the cluster core in the non-conservative Phe48 muta nts. Increased solvent access has been characterized by H-1-N-15 HMQC experiments.