Investigating the consequences of substituting histidine ligands to hemes in the decaheme cytochrome MtrC

Whiting, Keir (2024) Investigating the consequences of substituting histidine ligands to hemes in the decaheme cytochrome MtrC. Masters thesis, University of East Anglia.

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Abstract

MtrC is a decaheme c-type cytochrome found naturally on the external face of the outer membrane of Shewanella oneidensis MR-1. Previous studies have prepared a soluble version of this protein containing a surface tyrosine to cysteine (Y657C) substitution that can be photosensitised with a Ru(II)bipyridine3 dye. This surface mounted dye allowed for pump-probe spectroscopy experiments to measure electron transfer rates across MtrC. In the same experiments the substitution of a distal histidine ligand to heme eight with a methionine residue increased the lifetime of the charge separated state. This work discusses the production and characterisation of two additional heme distal ligand substitutions, a heme eight histidine to cysteine (H561C) substitution, and a heme three histidine to methionine (H230M) substitution. Electronic absorbance and magnetic circular dichroism spectroscopies revealed all hemes in solution are low-spin, consistent with octahedral coordination of all iron centres. X-ray crystallographic structures of the H230M variant confirmed the sulphur of methionine 230 to be the distal ligand to heme three. However, the crystal structure of the H561C variant revealed the distal ligand to heme eight is not the sulphur of cysteine 561, but a post translational covalent modification to the cysteine. This covalent modification is likely to represent the ligand in solution as liquid chromatography-mass spectrometry revealed a homogeneous population with a mass of +51 Da higher than anticipated. Protein film electrochemical measurements reveal that both the H230M and H561C variants exhibit shifts in the macroscopic redox potential of one or more redox centres in the proteins. These findings offer the opportunity to explore the impact on the charge separated lifetime of the photosensitised proteins during pumpprobe spectroscopy, as well as offering new ways to study electron transfer dynamics in multiheme cytochromes through introduction of unique spectroscopic centres.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024)
Depositing User:	Chris White
Date Deposited:	07 Jan 2025 09:53
Last Modified:	07 Jan 2025 09:53
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98112
DOI:

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