The R481 residue of cytochrome bo3 ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo3 from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [Puustinen, A. and Wikstr̈om, M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 35-37]. In an effort to delineate the structural role of R481 in the bo3 oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o 3 coordination state, reflecting a change in position and/or coordination of the CuB located on the distal side of heme o3, although it is ∼10 Å away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-Ostretching modes indicate that, unlike the wild type protein, the CuB is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the β conformer of the CO-adduct, indicating a shift in the equilibrium between the α and β conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the CuB site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.
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