Different pathways of radical translocation in yeast cytochrome c peroxidase and its W191F mutant on reaction with H(2)O(2) suggest an antioxidant role.
In the absence of exogenous donors, turnover of 10 molar equivalents of H(2)O(2) by wild-type recombinant cytochrome c peroxidase [CCP(MI)] and its W191F mutant at pH 7.0 occurs by oxidation of endogenous donors on the polypeptide. No O(2) evolution was observed with either enzyme on reaction with 10 molar equivalents of H(2)O(2), eliminating catalase-like activity, but O(2) evolution was observed when 100 molar equivalents of H(2)O(2) were added to the enzymes. Protein dimers were observed by SDS-PAGE following H(2)O(2) turnover by the peroxidases, and dimeric forms of CCP(MI) and CCP(W191) were isolated by gel-permeation chromatography. LC-ESI-MS analysis of the tryptic digests of the dimers revealed the previously reported T(6)-T(6) crosslink and a new crosslink between T(6)-T(26), but no T(26)-T(26) crosslink. The crosslinked tryptic peptides contain the exposed tyrosine residues Tyr36, Tyr39 and Tyr42 (T(6)), and Tyr229 and Tyr236 (T(26)). Addition of a spin trap, 2-methyl-2-nitrosopropane (MNP), to the CCP(MI)/H(2)O(2) reaction resulted in MNP labeling of peptides T(6), T(21) (which contains Tyr153) and T(26). MNP labeling of Tyr236 was found by sequencing peptide T(26). MNP labeling did not compete with dimerization of H(2)O(2)-oxidized CCP(W191F), suggesting that dityrosine formation in this mutant is very rapid owing to the high reactivity of radicals formed on T(6). H(2)O(2)-dependent formation of CCP-cytochrome c heterodimers was observed for both CCP(MI) and W191F in the presence of ferricytochrome c, the oxidized form of CCP's donor substrate. Interestingly, no H(2)O(2)-dependent cytochrome crosslinking to the W51F mutant was observed, even though this mutant underwent extensive homocrosslinking. The translocation of oxidizing equivalents from the heme to the surface residues of CCP is discussed in terms of an antioxidant role for CCP.
Tsaprailis
George
G
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
English
Ann M
AM
D019943:Amino Acid Substitution; D000975:Antioxidants / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005609:Free Radicals / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009603:Nitroso Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013113:Spin Labels; D014443:Tyrosine / Q000737:chemistry
D000975:Antioxidants; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D005609:Free Radicals; D005656:Fungal Proteins; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D013113:Spin Labels; D014443:Tyrosine; D006418:Heme; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase; C034526:tert-nitrosobutane; D010100:Oxygen