Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding.
Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.
Qian
J
J
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Shi
W
W
Pressler
M
M
Hoganson
C
C
Mills
D
D
Babcock
G T
GT
Ferguson-Miller
S
S
D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D013059:Spectrum Analysis, Raman
D017494:Proton Pumps; D001224:Aspartic Acid; D008345:Manganese; D003300:Copper; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV