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Title: Spectroscopic and electrochemical studies of di-heme thiosulphate dehydrogenases
Author: Jenner, Leon P.
ISNI:       0000 0004 7969 6855
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2019
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Thiosulphate (SSO32-) and tetrathionate (S4O62-) are found in environments where numerous bacterial species live. Some of these bacteria express di-heme thiosulphate dehydrogenase (TsdA) enzymes that catalyse the interconversion of these sulphur compounds: 2 SSO32- ↔ S4O62- + 2 e- (EM = +198 mV) Electrons originate or terminate in this redox couple during respiration or photosynthesis in these bacteria. This is likely to give them a niche competitive advantage against other organisms lacking this enzyme in environments where these compounds are present. The catalytic site of TsdA enzymes is heme I which has the unusual axial ligand pairing His/Cys-. Heme II is an electron transfer site which has His/Met ligation in most TsdA enzymes (eg. Campylobacter jejuni (Cj) TsdA), but which has a redox-driven switch between His/Lys and His/Met ligation in two known TsdA enzymes (eg. Allochromatium vinosum (Av) TsdA). This work uses magnetic circular dichroism (MCD) to identify the heme ligation states for recombinant TsdA enzymes Cj TsdA and Av TsdA in solution and several variants where these ligands are substituted. Protein film electrochemistry (PFE) also characterised the electroactivity of the TsdA hemes in these proteins. This, in combination with electrochemically-poised MCD samples, was used to determine the mid-point potentials of native and variant hemes. Additionaly these techniques confirmed the Av TsdA heme II ligand switch and permitted investigation of its kinetics. Remarkably, the number of electrons transferred by Cj TsdA heme I in PFE is different to the number transferred by heme II. In comparison, the number of electrons transferred by the Av TsdA hemes are equal, although introducing Cj TsdA-like heme II ligation, introduces unequal electron transfer. Replacing the heme I cysteine ligand with methionine in Cj TsdA causes both hemes to have equal electron transfer. Liquid chromatography mass spectrometry (LC-MS)-resolved native covalent adducts to the heme I cysteine ligand, which modulate the electroactivity of heme I. A catalytic mechanism proposed for TsdA enzymes features covalent modification of the heme I cysteine ligand. Chemical treatments, substrates and substrate analogues have been used to change these modifications in a predictable manner, allowing for the characterisation of TsdA with defined heme I cysteine modifications. Combining data from PFE, MCD and LC-MS the previously-proposed catalytic mechanism is expanded further. Evidence suggests that the substrate-augmented site has adducts to the heme I cysteine which also control electron transfer to the TsdA hemes. This electron transfer is not adequately explained by conventional theory of redox centres in proteins suggesting that the reality in TsdA enzymes is more complex than this typically assumed model.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available