title: Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site.
abstract: Time-resolved electron transfer and electrogenic H(+) translocation have been compared in a bd-type quinol oxidase from Escherichia coli and its E445A mutant. The high-spin heme b(595) is found to be retained by the enzyme in contrast to the original proposal, but it is not reducible even by excess of dithionite. When preincubated with the reductants, both the WT (b(558)(2+), b(595)(2+), d(2+)) and E445A mutant oxidase (b(558)(2+), b(595)(3+), d(2+)) bind O(2) rapidly, but formation of the oxoferryl state in the mutant is approximately 100-fold slower than in the WT enzyme. At the same time, the E445A substitution does not affect intraprotein electron re-equilibration after the photolysis of CO bound to ferrous heme d in the one-electron-reduced enzyme (the so-called "electron backflow"). The backflow is coupled to membrane potential generation. Electron transfer between hemes d and b(558) is electrogenic. In contrast, electron transfer between hemes d and b(595) is not electrogenic, although heme b(595) is the major electron acceptor for heme d during the backflow, and therefore is not likely to be accompanied by net H(+) uptake or release. The E445A replacement does not alter electron distribution between hemes b(595) and d in the one-electron reduced cytochrome bd [E(m)(d) > E(m)(b(595)), where E(m) is the midpoint redox potential]; however, it precludes reduction of heme b(595), given heme d has been reduced already by the first electron. Presumably, E445 is one of the two redox-linked ionizable groups required for charge compensation of the di-heme oxygen-reducing site (b(595), d) upon its full reduction by two electrons.
authors.affiliation: Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FIN-00014 Helsinki, Finland.
authors.forename: Ilya
authors.initials: I
authors.lastname: Belevich
authors.forename: Vitaliy B
authors.initials: VB
authors.lastname: Borisov
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Ke
authors.initials: K
authors.lastname: Yang
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Konstantinov
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Michael I
authors.initials: MI
authors.lastname: Verkhovsky
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000502:physiology; D008564:Membrane Potentials; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; C435883:heme b(595); D004227:Dithionite; D006418:Heme; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli
references.citation: FEBS Lett. 1993 Aug 2;327(3):279-83
references.pmid: 8348954
references.citation: Chem Rev. 1996 Nov 7;96(7):2889-2908
references.pmid: 11848844
references.citation: Biochim Biophys Acta. 2003 Aug 18;1605(1-3):1-13
references.pmid: 12907296
references.citation: Biochemistry. 1999 Jan 12;38(2):740-50
references.pmid: 9888814
references.citation: J Biol Chem. 1985 Nov 15;260(26):14003-8
references.pmid: 2414286
references.citation: FEBS Lett. 1997 Sep 8;414(2):369-72
references.pmid: 9315721
references.citation: Biochemistry. 1991 Dec 10;30(49):11485-9
references.pmid: 1747367
references.citation: Biochim Biophys Acta. 1984 Oct 23;790(2):148-53
references.pmid: 6386051
references.citation: Biokhimiia. 1996 May;61(5):786-99
references.pmid: 8754266
references.citation: Biochemistry. 2002 Feb 5;41(5):1654-62
references.pmid: 11814360
references.citation: J Bacteriol. 1997 Jun;179(11):3813-7
references.pmid: 9171438
references.citation: FEBS Lett. 2004 Mar 12;561(1-3):58-62
references.pmid: 15013751
references.citation: Biochemistry. 1999 Apr 13;38(15):4853-61
references.pmid: 10200174
references.citation: Biochemistry. 1991 Apr 23;30(16):3936-42
references.pmid: 1850294
references.citation: J Inorg Biochem. 2002 Jul 25;91(1):87-93
references.pmid: 12121765
references.citation: Adv Microb Physiol. 2000;43:165-224
references.pmid: 10907557
references.citation: Biochemistry. 1984 Jan 31;23(3):445-53
references.pmid: 6367818
references.citation: Biochemistry. 2001 Jul 24;40(29):8548-56
references.pmid: 11456494
references.citation: Biochim Biophys Acta. 1999 Jan 27;1410(1):32-50
references.pmid: 10076013
references.citation: Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9085-90
references.pmid: 9256439
references.citation: Biochim Biophys Acta. 1987 Sep 10;893(2):289-95
references.pmid: 3040093
references.citation: Biochemistry. 1994 Dec 20;33(50):15110-5
references.pmid: 7999770
references.citation: J Biol Chem. 2001 Jun 22;276(25):22095-9
references.pmid: 11283005
references.citation: Nature. 2004 Jan 29;427(6973):441-4
references.pmid: 14749831
references.citation: J Biol Chem. 1994 Jan 28;269(4):2419-25
references.pmid: 8300568
references.citation: FEBS Lett. 1987 Jun 8;217(1):49-52
references.pmid: 3036575
references.citation: Biochemistry. 1997 Aug 5;36(31):9323-31
references.pmid: 9235974
references.citation: J Biol Chem. 1995 Dec 1;270(48):28565-9
references.pmid: 7499371
references.citation: Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5863-7
references.pmid: 8516338
references.citation: Annu Rev Biochem. 1994;63:675-716
references.pmid: 7979252
references.citation: Biochemistry. 2000 Nov 14;39(45):13800-9
references.pmid: 11076519
references.citation: Nature. 2002 May 2;417(6884):99-102
references.pmid: 11986672
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1554-9
references.pmid: 10660685
references.citation: Anal Biochem. 1987 Feb 15;161(1):1-15
references.pmid: 3578775
references.citation: Biochim Biophys Acta. 1997 Aug 22;1321(2):107-27
references.pmid: 9332500
references.citation: Physiol Rev. 1970 Jul;50(3):319-75
references.pmid: 4912904
references.citation: J Biol Chem. 1984 Mar 10;259(5):3375-81
references.pmid: 6321507
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):529-33
references.pmid: 14699047
references.citation: Biochemistry. 1999 May 4;38(18):5913-7
references.pmid: 10231544
references.citation: Biochemistry. 2001 Jun 12;40(23):6882-92
references.pmid: 11389603
references.citation: Methods Enzymol. 1986;126:87-94
references.pmid: 2856143
references.citation: Biochemistry. 1999 Mar 2;38(9):2697-706
references.pmid: 10052940
references.citation: Biochemistry. 1986 May 6;25(9):2314-21
references.pmid: 3013299