A LOOP BETWEEN TRANSMEMBRANE HELIX-IX AND HELIX-X OF SUBUNIT-I OF CYTOCHROME-C-OXIDASE CAPS THE HEME ALPHA-HEME ALPHA(3)-CU-B CENTER

Site-directed mutants were prepared of four consecutive and highly conserved residues (His-411 1, Asp-412, Thr-413, Tyr-414) of an extramembrane leap that connects putative transmembrane helices IX and X of subunit I of Rhodobacter sphaeroides cytochrome c oxidase. The modified enzymes were purified and analyzed by optical, resonance Raman, FTIR, and EPR spectroscopies. Consistent with our recent model in which both hemes are ligated to histidines of helix X [Hosler, J. P., et al. (1993) J. Bioenerg. Biomembr. 25, 121-136], substitutions for three of these four residues cause perturbations of either heme alpha or heme alpha(3). Resonance Raman spectra of the mutant Y414F demonstrate that Tyr-414 does not participate in a hydrogen bond with the heme alpha formyl group, but its alteration does result in a 5-nm red-shift of the alpha-band of the visible spectrum, indicating proximity to heme alpha. The mutant D412N shows changes in resonance Raman and FTIR difference spectra indicative of an effect on the proximal ligation of heme alpha 3 Changing His-411 to alanine has relatively minor effects on the spectral and functional properties of the oxidase; however, FTIR spectra reveal alterations in the environment of Cu-B. Conversion of this residue to asparagine strongly disrupts the environment of heme alpha 3 and CUB and inactivates the enzyme. These results suggest that His-411 is very near the heme alpha 3-Cu-B pocket. We propose that these residues form part of a cap over the heme alpha-heme alpha(3)-Cu-B center and thus are important in the structure of the active site.