Fluorescence quenching of reconstituted NCD-4-labeled cytochrome c oxidase complex by DOXYL-stearic acids.
It has been known for some time that dicyclohexylcarbodiimide (DCCD) inhibits the proton translocation function of the cytochrome c oxidase complex (CcO) and that there is one major site in subunit III which is modified upon reaction with DCCD (Glu-90 for the bovine enzyme). We have examined the reaction of bovine CcO with N-cyclohexyl-N'-(4-dimethylamino-alpha-napthyl)carbodiimide (NCD-4), a fluorescent analog of DCCD. NCD-4 labeling of CcO is strongly inhibited by DCCD implicating Glu-90 of subunit III as the site of chemical modification by NCD-4. The fluorescence of reconstituted NCD-4-labeled bovine CcO is strongly quenched by hydrophobic nitroxides, whereas hydrophilic nitroxides and iodide ions have a reduced quenching ability. It is concluded that the Glu-90 of subunit III resides near the protein-lipid interface of the membrane spanning region of the enzyme. Different quenching abilities of 5-, 7-, 10-, 12-, and 16-4,4-dimethyl-3-oxazolinyloxy-stearic acids suggest that the NCD-4 label is located in the membrane bilayer in the region near the middle of the hydrocarbon tail of stearic acid. In light of these results, it is unlikely that Glu-90 is part of a proton channel that is associated with the proton pumping machinery of the enzyme but the outcome of this study does not eliminate an allosteric regulatory role for this residue.
Musser
S M
SM
Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Larsen
R W
RW
Chan
S I
SI
D000595:Amino Acid Sequence; D000818:Animals; D002234:Carbodiimides; D002417:Cattle; D003497:Cyclic N-Oxides; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry / Q000379:methods; D013113:Spin Labels
D002234:Carbodiimides; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013113:Spin Labels; C037404:12-doxylstearic acid; C032363:16-nitroxystearic acid; D004024:Dicyclohexylcarbodiimide; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D003576:Electron Transport Complex IV