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

Abstract

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.

Figure 1

Resonance Raman spectra of the oxidized forms of the wt and R481 mutants of RsCcO. The identities of the spectra and the associated difference spectra are as indicated. Right Panel: Lorentzian curve-fitting of the difference spectrum (e); the positive and negative bands are attributed to R481H and wt, respectively. For the fitting, the intensities, frequencies and widths were free-floating. The frequencies from the best-fit are listed in Table 1. The excitation wavelength was 413.1 nm.

Figure 2

Resonance Raman spectra of the reduced forms of the wt and R481 mutants of RsCcO. The identities of the spectra and the associated difference spectra are as indicated. Right Panel: Lorentzian curve-fitting of the difference spectrum (e); the positive and negative bands are attributed to R481H and wt, respectively. For the fitting, the intensities, frequencies and widths were free-floating. The frequencies from the best-fit are listed in Table 1. The excitation wavelength was 413.1 nm.

Figure 3

The extended hydrogen bonding network in the catalytic site of RsCcO (PDB: 1M56). The two hemes, their formyl groups and the critical residues are as indicated. The two red spheres represent water molecules. The figure was rendered with PyMOL.