Critical structural role of R481 in cytochrome c oxidase from Rhodobacter sphaeroides

The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a(3). It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to approximately 5-18% of the native level, and the absence of proton pumping in R481Q but retention of approximately 40% efficiency in R481H and R481L (H.J. Lee, L. Ojemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl CO stretching modes of both the heme a and a(3) in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a(3). In addition to the change in the environments of heme a and a(3), the Raman data show that the mutations induce a partial conversion of the heme a(3) from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the Cu(B)-heme a(3) binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.