A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

Mary Ortmayer,Florence J Hardy,Colin Levy,Anthony P Green,Sam Hay,Karl Fisher,Stephen E J Rigby,Derren J Heyes,Sam P De Visser,Matthew G Quesne,C Richard A Catlow

doi:10.1021/jacsau.1c00145

Mary Ortmayer, Florence J Hardy + Show 9 more

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https://doi.org/10.1021/jacsau.1c00145

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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 c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-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.

Highlights

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity
This challenge is acute when probing the role of the largest canonical amino acid, tryptophan, which has no close structural analogue within the genetic code
The availability of an expanded alphabet of amino acids provides a more surgical means of probing biological mechanisms by allowing substitutions of individual atoms or functional groups within proteins of interest.[2−6] The power of this approach is exemplified through recent studies, whereby noncanonical cysteine and histidine analogues have been used to examine the role of axial heme ligands in controlling the reactivities of iconic ferryl intermediates compound I and compound II.[7−13] These high-energy intermediates are the defining feature that drive catalysis across the entire family of heme enzymes, including P450s, peroxidases, nitric oxide synthases, and terminal oxidases.[14,15]

Summary

Corresponding Author

Hardy − Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, United Kingdom; orcid.org/00000003-0671-0209. Quesne − Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxon OX11 0FA, United Kingdom; Cardiff University, School of Chemistry, Park Place, Cardiff CF10 3AT, United Kingdom; orcid.org/0000-0001-5130-1266. Catlow − Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxon OX11 0FA, United Kingdom; Cardiff University, School of Chemistry, Park Place, Cardiff CF10 3AT, United Kingdom; Kathleen Lonsdale Materials Chemistry, Department of Chemistry, University College London, London, Western Central 1H 0AJ, United Kingdom Sam P. de Visser − Department of Chemical Engineering and Analytical Science & Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, United Kingdom; orcid.org/0000-0002-2620-8788 Stephen E.

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