title: Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.
abstract: Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.
authors.affiliation: Department of Molecular Biology, University of California at Irvine, 92717, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Pappa
authors.forename: S
authors.initials: S
authors.lastname: Tajbaksh
authors.forename: A J
authors.initials: AJ
authors.lastname: Saunders
authors.forename: G J
authors.initials: GJ
authors.lastname: Pielak
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006736:Horses; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D015345:Oligonucleotide Probes; D003578:Cytochrome-c Peroxidase