6Y2Y

The crystal structure of engineered cytochrome c peroxidase from Saccharomyces cerevisiae with Trp51 to S-Trp51 and Trp191Phe modifications

Summary for 6Y2Y

• Entry DOI: 10.2210/pdb6y2y/pdb
• Descriptor: Cytochrome c peroxidase, mitochondrial, PROTOPORPHYRIN IX CONTAINING FE, 1,2-ETHANEDIOL, ... (4 entities in total)
• Functional Keywords: peroxidase, heme, engineered, non-canonical amino acid, oxidoreductase
• Biological source: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast)
• Total number of polymer chains: 1
• Total formula weight: 37587.45

Authors

Ortmayer, M.,Levy, C.,Green, A.P. (deposition date: 2020-02-17, release date: 2021-06-16, Last modification date: 2024-11-06)

Primary citation

Ortmayer, M.,Hardy, F.J.,Quesne, M.G.,Fisher, K.,Levy, C.,Heyes, D.J.,Catlow, C.R.A.,de Visser, S.P.,Rigby, S.E.J.,Hay, S.,Green, A.P.
A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.
Jacs Au, 1:913-918, 2021

PubMed Abstract: Nature 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 peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CP 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 cyt. 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.

PubMed: 34337604
DOI: 10.1021/jacsau.1c00145

Experimental method

X-RAY DIFFRACTION (1.7 Å)