Site-directed mutants were prepared of four consecutive and highly conserved residues (His-411, Asp-412, Thr-413, Tyr-414) of an extramembrane loop 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 a or heme a3. Resonance Raman spectra of the mutant Y414F demonstrate that Tyr-414 does not participate in a hydrogen bond with the heme a formyl group, but its alteration does result in a 5-nm red-shift of the α-band of the visible spectrum, indicating proximity to heme a. The mutant D412N shows changes in resonance Raman and FTIR difference spectra indicative of an effect on the proximal ligation of heme a3. 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 CuB. Conversion of this residue to asparagine strongly disrupts the environment of heme a3 and CuB and inactivates the enzyme. These results suggest that His-411 is very near the heme a3-CuB pocket. We propose that these residues form part of a cap over the heme a-heme a3-CuB center and thus are important in the structure of the active site.