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 con served residues (His-411 1, Asp-412, Thr-413, Tyr-414) of an extramemb rane leap that connects putative transmembrane helices IX and X of sub unit I of Rhodobacter sphaeroides cytochrome c oxidase. The modified e nzymes were purified and analyzed by optical, resonance Raman, FTIR, a nd EPR spectroscopies. Consistent with our recent model in which both hemes are ligated to histidines of helix X [Hosler, J. P., et al. (199 3) J. Bioenerg. Biomembr. 25, 121-136], substitutions for three of the se four residues cause perturbations of either heme alpha or heme alph a(3). Resonance Raman spectra of the mutant Y414F demonstrate that Tyr -414 does not participate in a hydrogen bond with the heme alpha formy l group, but its alteration does result in a 5-nm red-shift of the alp ha-band of the visible spectrum, indicating proximity to heme alpha. T he mutant D412N shows changes in resonance Raman and FTIR difference s pectra indicative of an effect on the proximal ligation of heme alpha 3 Changing His-411 to alanine has relatively minor effects on the spec tral and functional properties of the oxidase; however, FTIR spectra r eveal alterations in the environment of Cu-B. Conversion of this resid ue 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 residue s 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.