title: Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.
abstract: Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
authors.forename: J S
authors.initials: JS
authors.lastname: Felsch
authors.forename: M P
authors.initials: MP
authors.lastname: Horvath
authors.forename: S
authors.initials: S
authors.lastname: Gursky
authors.forename: M R
authors.initials: MR
authors.lastname: Hobaugh
authors.forename: P N
authors.initials: PN
authors.lastname: Goudreau
authors.forename: J A
authors.initials: JA
authors.lastname: Fee
authors.forename: W T
authors.initials: WT
authors.lastname: Morgan
authors.forename: S J
authors.initials: SJ
authors.lastname: Admiraal
authors.forename: M
authors.initials: M
authors.lastname: Ikeda-Saito
authors.forename: T
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002417:Cattle; D017936:Cytochrome a Group / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006466:Hemopexin / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods; D014508:Urea / Q000494:pharmacology; D000099053:Histidine-Rich Glycoprotein
chemical_list: D001059:Apoproteins; D017936:Cytochrome a Group; D009211:Myoglobin; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; C027728:heme a; D006418:Heme; D014508:Urea; D006466:Hemopexin; D003576:Electron Transport Complex IV