A Novel Heme and Peroxide-dependent Tryptophan–tyrosine Cross-link in a Mutant of Cytochrome c Peroxidase

Abstract

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to a Tyr reveals that the tyrosine side chain forms a covalent bond with the indole ring nitrogen of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 crosslink. Instead, the Tyr52 side chain orients away from the heme active site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron. 3 KeywordsCytochrome c peroxidase; X-ray crystal structure; Fe-containing heme; Zn-containing protoporphyrin IX; oxygen radical; tryptophan-tyrosine cross-link; Trp cation radical. 4 AbbreviationsCcP, cytochrome c peroxidase; CcO, cytochrome c oxidase; cyt. c, reduced horse heart or yeast cytochrome c; EDTA, ethylenediaminetetraacetic acid; MPD, 2-methyl-2,4-pentanediol; KPB, potassium phosphate buffer; M r , relative molecular mass; IPTG, isopropylthio--galactoside 5

Radical Sites in Mycobacterium tuberculosis KatG Identified Using Electron Paramagnetic Resonance Spectroscopy, the Three-dimensional Crystal Structure, and Electron Transfer Couplings

“…The discrepancy may be explained by the fact that storage of the enzyme and the low pH conditions required for crystallization both may favor adduct formation. Heme catalysis is required for this processing and during growth, isolation and purification, and storage of KatG will depend directly on the level of endogenous peroxides available to initiate heme turnover (6,7,17).…”

“…(Trp 51 , conserved in all Class I peroxidases, is conserved at position 107 in Mtb KatG.) The reaction leading to that modification has also been shown to require turnover of the enzyme and presumably, radical recombination at these sites (17).…”

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

“…Peroxidase activity has been shown to mediate cross-linking of free indole groups [41,42]. A tryptophan tyrosine cross link could be formed as a result of peroxidase activity of a mutant form of yeast cytochrome c peroxidase [43]. Intriguingly, a distal histidine mutation in the same enzyme catalyses hydrogen peroxide oxidation rather than reduction, and may generate a labile superoxide [44].…”

The Met-Tyr-Trp Cross-link in Mycobacterium tuberculosis Catalase-peroxidase (KatG)

“…In the only other example of a Tyr-Trp bond, CcP(H52Y) (73), the histidine-to-tyrosine mutation allows for the formation of a C-N bond (as opposed to the C-C bond in M. tuberculosis KatG) between the indolic-N (N⑀1) of Trp 51 and the C⑀1 of Tyr 52 . Poulos and coworkers (73) argue that, given the short lifetimes of protein radicals, two sequential one-electron oxidations by two compound I intermediates (resulting from two enzyme turnovers) yielding the Tyr-Trp bond is unlikely to occur in CcP(H52Y). Rather, they argue that any mechanism for bond formation between two protein side chains must support concurrent radical formation on both protein residues, and our data, which demonstrate the lack of CLPF formation derived from KatG compound II (one oxidizing equivalent above the resting state), supports this notion.…”