Photooxidation of Trp-191 in cytochrome c peroxidase by ruthenium-cytochrome c derivatives.
A novel photoinduced electron-transfer reaction is reported in complexes between resting ferric state cytochrome c peroxidase (CcP) and several horse cytochrome c derivatives labeled at single lysine amino groups with [bis(bipyridine)](dicarboxybipyridine)ruthenium(II) (Ru-CC). Photoexcitation of Ru(II) in the 1:1 Ru-27-CC:CcP complex results in formation of a metal-to-ligand charge-transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to the CC heme Fe(III) with rate constant k1 = 2.3 x 10(7) s-1. The resulting Ru(III) is a strong oxidizing agent with a redox potential of 1.3 V, and it oxidizes the indole ring of Trp-191 with rate constant k3 = 7 x 10(6) s-1. The cycle is completed by electron transfer from Fe(II) in CC to the Trp-191 radical in CcP with rate constant k4 = 6.1 x 10(4) s-1. The Ru group is located close to the interaction domain in the Ru-27-CC:CcP complex, allowing rapid electron transfer with both the heme in CC and Trp-191 in CcP. The electron-transfer reaction was not observed in CcP compound I, where Trp-191 is already oxidized to the radical, or in the W191F mutant, where the indole group is replaced with a phenyl group. The electron-transfer reaction was observed in CcP mutants modified at residues in the heme crevice, R48K, R48L, H52L, M230I, and M231I, but not in D235N which destabilizes the radical on Trp-191. Increasing the ionic strength results in an increase in the equilibrium dissociation constant K of the Ru-27-CC:CcP complex and an increase in the rate constant k5 for dissociation of the transient intermediate containing Fe(II) CC and the radical form of CcP. Both K and k5 were also increased significantly by the mutations D34N, E290N, and A193F involving residues located in the interaction domain of the crystalline complex between yeast CC and CcP [Pelletier & Kraut (1992) Science 258, 1748-1755]. This new method allows the study of the electron-transfer reaction between CC and the radical on Trp-191 in the complete absence of hydrogen peroxide, and it opens the possibility of measurements at low temperatures in frozen glasses or in crystals.
Liu
R Q
RQ
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Hahm
S
S
Miller
M
M
Durham
B
B
Millett
F
F
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D006736:Horses; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010777:Photochemistry; D010782:Photolysis; D017434:Protein Structure, Tertiary; D012428:Ruthenium / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D003574:Cytochrome c Group; D012428:Ruthenium; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase