title: Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: I. Spectral and functional properties.
abstract: To study the functional significance of the unusual bimetallic Cu(A) center of cytochrome c oxidase, the direct ligands of the Cu(A) center in subunit II of the holoenzyme were mutated. Two of the mutant forms, M263L and H260N, exhibit major changes in activity (10% and 1% of wild-type, respectively) and in near-infrared and EPR spectra, but metal analysis shows that both mutants retain two coppers in the Cu(A) center and both retain proton pumping activity. In M263L, multifrequency EPR studies indicate the coppers are still electronically coupled, while all the other metal centers in M263L appear unchanged, by visible, EPR, and FTIR spectroscopy. Nevertheless, heme a3 is very slow to reduce with cytochrome c or dithionite under stopped-flow and steady-state conditions. This effect appears to be secondary to the change in redox equilibrium between Cu(A) and heme a. The studies reported here and in Wang et al. [Wang, K., Geren, L., Zhen, Y., Ma, L., Ferguson-Miller, S., Durham, B., and Millett, F. (2002) Biochemistry 41, 2298-2304] demonstrate that altering the ligands of Cu(A) can influence the rate and equilibrium of electron transfer between Cu(A) and heme a, but that the native ligation state is not essential for proton pumping.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Zhen
authors.forename: Bryan
authors.initials: B
authors.lastname: Schmidt
authors.forename: Uan Gen
authors.initials: UG
authors.lastname: Kang
authors.forename: William
authors.initials: W
authors.lastname: Antholine
authors.forename: Shelagh
authors.initials: S
authors.lastname: Ferguson-Miller
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D006639:Histidine / Q000235:genetics; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008345:Manganese / Q000737:chemistry; D008715:Methionine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary / Q000235:genetics; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D017494:Proton Pumps; C027728:heme a; D006418:Heme; D008345:Manganese; D006639:Histidine; D001216:Asparagine; D003300:Copper; D008715:Methionine; D007501:Iron; D003576:Electron Transport Complex IV; D007930:Leucine; D005998:Glycine