Resonance Raman and Fourier transform infrared studies on the subunit I histidine mutants of the cytochrome bo complex in Escherichia coli. Molecular structure of redox metal centers.

Abstract

The cytochrome bo complex is a heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. Site-directed mutagenesis studies were greatly successful in identifying ligands of the redox centers in the oxidase (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). In the present study, resonance Raman and Fourier transform infrared spectroscopies were applied to the subunit I histidine mutant oxidases, and the active-site structure was studied in detail. In the dithionite-reduced state, the wild-type oxidase and the His-284-->Ala and His-333-->Ala mutant oxidases showed two Raman lines (v3) at approximately 1492 and 1472 cm-1, which are attributable to low- and high-spin heme components, respectively. The Ala substitutions at His-106 and His-421 reduced the low-spin line, whereas the His-419-->Ala mutation eliminated the high-spin line. This indicates that His-106 and His-421 are the axial ligands of the low-spin heme B, while His-419 acts as a proximal ligand of the high-spin heme O. The v(Fe-CO) stretching frequency in the wild-type oxidase down-shifted from 523 to 498 cm-1 in the CuB-deficient mutant oxidase (His-333-->Ala), while the v(C-O) stretching frequency up-shifted from 1960 to 1970 cm-1. These frequency differences were ascribed to the contribution from the reduced CuB at the binuclear center. It may indicate that delocalization of electrons at the CuB center toward the heme O iron is facilitated by a bridge ligand structure at the binuclear center and is essential for driving dioxygen reduction chemistry, and therefore, redox-coupled proton pumping.