CHARACTERIZATION OF AN AUTOREDUCTION PATHWAY FOR THE [FE4S4](3-VINOSUM HIGH-POTENTIAL IRON PROTEINS - SITE-DIRECTED MUTAGENESIS STUDIES TO PROBE THE ROLE OF PHENYLALANINE 66 IN DEFINING THE STABILITY OF THE [FE4S4] CENTER PROVIDE EVIDENCE FOR OXIDATIVE-DEGRADATION VIA A [FE3S4] CLUSTER() CLUSTER OF MUTANT CHROMATIUM)

A number of point mutations of the conserved aromatic residue phenylal anine 66 (Phe66Tyr, -Asn, -Cys, -Ser) in Chromatium vinosum high-poten tial iron sulfur protein have been examined with the aim of understand ing the functional role of this residue, Nonconservative replacements with polar residues have a minimal effect on the midpoint potential of the [Fe4S4](3+/2+) cluster, typically <+25 mV, with a maximum change of +40 MV for Phe66Asn. With the exception of the Phe66Tyr mutant. the oxidized slate was found to be unstable relative to the recombinant n ative, with regeneration of the reduced state. The pathway for this tr ansformation involves degradation of the cluster in a fraction of the sample, which provides the reducing equivalents required to bring abou t reduction of the remainder of tile sample. This degradative reaction proceeds through a transient [Fe3S4](+) intermediate that is characte rized by typical g values and power saturation behavior and is prompte d by the increased solvent accessibility of the cluster core in the no nconservative Phe66 mutants as evidenced by H-1-N-15 HMQC NMR experime nts. These results are consistent with a model where the critical role of the aromatic residues in the high-potential iron proteins is to pr otect the cluster from hydrolytic degradation in the oxidized states.