Rewiring the ‘push-pull’ catalytic machinery of a heme enzyme using an expanded genetic code

DOI: 10.1021/acscatal.9b05129

Authors: Mary Ortmayer (Manchester Institute of Biotechnology, University of Manchester) , Karl Fisher (Manchester Institute of Biotechnology, University of Manchester) , Jaswir Basran (University of Leicester) , Emmanuel M. Wolde-Michael (Manchester Institute of Biotechnology, University of Manchester) , Derren J. Heyes (Manchester Institute of Biotechnology, University of Manchester) , Colin Levy (Manchester Institute of Biotechnology, University of Manchester) , Sarah Lovelock (Manchester Institute of Biotechnology, University of Manchester) , J. L. Ross Anderson (University of Bristol) , Emma L. Raven (University of Bristol) , Sam Hay (Manchester Institute of Biotechnology, University of Manchester) , Stephen E. J. Rigby (Manchester Institute of Biotechnology, University of Manchester) , Anthony P. Green (Manchester Institute of Biotechnology, University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: January 2020
Diamond Proposal Number(s): 12788

Abstract: Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron donating Nδ-methyl histidine (Me-His) with little effect on enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron do-nation and compound II reactivity which can be explained by weaker electron donation from the Me-His ligand (‘the push’) affording an electron deficient ferryl-oxygen with reduced proton affinity (‘the pull’). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation, designed to increase ‘the pull’ by removing a hydrogen bond to the ferryl-oxygen. Analogous substitutions in ascorbate peroxidase (APX) lead to similar activity trends to those observed in CcP, suggesting a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how non-canonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

Journal Keywords: Heme enzyme; non-canonical ligand; metal-oxo reactivity; proton coupled electron transfer

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Added On: 03/02/2020 10:25

Documents:
7fgh5.pdf

Discipline Tags:

Biochemistry Genetics Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)