Roles of noncoordinated aromatic residues in redox regulation of cytochrome c3 from Desulfovibrio vulgaris Miyazaki F.

The roles of aromatic residues in redox regulation of cytochrome c(3) were investigated by site-directed mutagenesis at every aromatic residue except for axial ligands (Phe20, Tyr43, Tyr65, Tyr66, His67, and Phe76). The mutations at Phe20 induced large chemical shift changes in the NMR signals for hemes 1 and 3, and large changes in the microscopic redox potentials of hemes 1 and 3. The NMR signals of the axial ligands of hemes 1 and 3 were also affected. Analysis of the nature of the mutations revealed that a hydrophobic environment and aromaticity are important for the reduction of the redox potentials of hemes 1 and 3, respectively. There was also a global effect. The replacement of Tyr66 with leucine induced chemical shift changes for heme 4, and changes in the microscopic redox potentials of heme 4. The mutations of Tyr65 induced changes in the chemical shifts and microscopic redox potentials for every heme, suggesting that Tyr65 stabilizes the global conformation, thereby reducing the redox potentials. In contrast, although the mutations of His67 and Phe76 caused chemical shift changes for heme 2, they did not affect its redox potentials, showing these residues are not important. All noncoordinated aromatic residues conserved in the cytochrome c(3) subfamily with heme binding motifs CXXCH, CXXXXCH, CXXCH, and CXXXXCH (Phe20, Tyr43, and Tyr66) are involved in the pi-pi interaction, which causes a decrease in the redox potential of the interacting heme. The global effect can be attributed to either direct or indirect interactions among the four hemes in the cyclic architecture.