title: Control of cyclic photoinitiated electron transfer between cytochrome c peroxidase (W191F) and cytochrome c by formation of dynamic binary and ternary complexes.
abstract: Extensive studies of the physiological protein-protein electron-transfer (ET) complex between yeast cytochrome c peroxidase (CcP) and cytochrome c (Cc) have left unresolved questions about how formation and dissociation of binary and ternary complexes influence ET. We probe this issue through a study of the photocycle of ET between Zn-protoporphyrin IX-substituted CcP(W191F) (ZnPCcP) and Cc. Photoexcitation of ZnPCcP in complex with Fe(3+)Cc initiates the photocycle: charge-separation ET, [(3)ZnPCcP, Fe(3+)Cc] → [ZnP(+)CcP, Fe(2+)Cc], followed by charge recombination, [ZnP(+)CcP, Fe(2+)Cc] → [ZnPCcP, Fe(3+)Cc]. The W191F mutation eliminates fast hole hopping through W191, enhancing accumulation of the charge-separated intermediate and extending the time scale for binding and dissociation of the charge-separated complex. Both triplet quenching and the charge-separated intermediate were monitored during titrations of ZnPCcP with Fe(3+)Cc, Fe(2+)Cc, and redox-inert CuCc. The results require a photocycle that includes dissociation and/or recombination of the charge-separated binary complex and a charge-separated ternary complex, [ZnP(+)CcP, Fe(2+)Cc, Fe(3+)Cc]. The expanded kinetic scheme formalizes earlier proposals of "substrate-assisted product dissociation" within the photocycle. The measurements yield the thermodynamic affinity constants for binding the first and second Cc: KI = 10(-7) M(-1), and KII = 10(-4) M(-1). However, two-site analysis of the thermodynamics of formation of the ternary complex reveals that Cc binds at the weaker-binding site with much greater affinity than previously recognized and places upper bounds on the contributions of repulsion between the two Cc's of the ternary complex. In conjunction with recent nuclear magnetic resonance studies, the analysis further suggests a dynamic view of the ternary complex, wherein neither Cc necessarily faithfully adopts the crystal-structure configuration because of Cc-Cc repulsion.
authors: Department of Chemistry, Northwestern University , 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors: Taylor R
authors: TR
authors: Page
authors: Brian M
authors: BM
authors: Hoffman
mesh_terms: D019943:Amino Acid Substitution; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D003578:Cytochrome-c Peroxidase; C526453:Cox1 protein, S cerevisiae; D003576:Electron Transport Complex IV
references: J Biol Chem. 1987 Jun 5;262(16):7719-24
references: 3034903
references: Biochemistry. 1999 May 25;38(21):6846-54
references: 10346906
references: Biochemistry. 2002 Mar 26;41(12):3968-76
references: 11900539
references: Eur J Biochem. 1986 Mar 17;155(3):505-11
references: 3007133
references: Biochemistry. 1990 Aug 7;29(31):7160-73
references: 2169873
references: Biochim Biophys Acta. 1987 Mar 18;912(1):87-97
references: 3030433
references: Science. 1995 Jul 14;269(5221):204-7
references: 7618081
references: Biochemistry. 2006 Dec 5;45(48):14371-8
references: 17128976
references: J Biomol NMR. 2010 Dec;48(4):225-36
references: 21049303
references: Biochemistry. 1996 Jan 23;35(3):667-73
references: 8547245
references: Science. 1988 Jul 1;241(4861):67-70
references: 2838904
references: Biochim Biophys Acta. 2011 Nov;1807(11):1482-503
references: 21820401
references: Biochemistry. 2000 Aug 22;39(33):10132-9
references: 10956001
references: Biochemistry. 1992 Apr 14;31(14):3661-70
references: 1314646
references: J Am Chem Soc. 2010 Jan 13;132(1):241-7
references: 19961227
references: Biochemistry. 1996 Dec 10;35(49):15800-6
references: 8961943
references: Chem Rev. 1996 Nov 7;96(7):2459-2490
references: 11848833
references: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5940-5
references: 15071191
references: Protein Expr Purif. 2000 Jun;19(1):139-47
references: 10833401
references: J Am Chem Soc. 1977 Jul 20;99(15):5168-73
references: 194941
references: Q Rev Biophys. 1968 May;1(1):35-80
references: 4885732
references: J Biol Chem. 1983 Apr 10;258(7):4356-63
references: 6300084
references: Biochemistry. 1994 Oct 25;33(42):12609-14
references: 7918486
references: Biochemistry. 1993 Oct 12;32(40):10848-65
references: 8399235
references: Biochemistry. 1992 Mar 3;31(8):2384-92
references: 1311599
references: Proc Natl Acad Sci U S A. 2006 Dec 12;103(50):18945-50
references: 17146057
references: Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15465-70
references: 16227441
references: J Biol Chem. 1967 Nov 10;242(21):5008-13
references: 6058943
references: Science. 1994 Sep 16;265(5179):1693-6
references: 8085152
references: Science. 1992 Dec 11;258(5089):1748-55
references: 1334573
references: J Am Chem Soc. 2005 Apr 27;127(16):5750-1
references: 15839648
references: J Biol Chem. 1977 Feb 10;252(3):919-26
references: 14138
references: Biochemistry. 1998 Apr 28;37(17):6124-31
references: 9558351