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The crystal structure of engineered cytochrome c peroxidase from Saccharomyces cerevisiae with a Trp51 to S-Trp51 modificationThe crystal structure of engineered cytochrome c peroxidase from Saccharomyces cerevisiae with a Trp51 to S-Trp51 modification
Structural highlights
FunctionCCPR_YEAST Destroys radicals which are normally produced within the cells and which are toxic to biological systems. Publication Abstract from PubMedNature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin pi-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity. A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.,Ortmayer M, Hardy FJ, Quesne MG, Fisher K, Levy C, Heyes DJ, Catlow CRA, de Visser SP, Rigby SEJ, Hay S, Green AP JACS Au. 2021 Jul 26;1(7):913-918. doi: 10.1021/jacsau.1c00145. Epub 2021 May 14. PMID:34337604[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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