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Title: Mechanistic characterisation of flavocytochrome c3
Author: Doherty, M. K.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Flavocytochrome c3 is a respiratory fumarate reductase produced by the marine organism Shewanella frigidimarina. It is a soluble enzyme located in the periplasm. The enzyme is composed of three domains; a flavin-binding domain, a cytochrome domain and a clamp domain. The flavin domain (residues 108-364 and 503-571) contains a non-covalently bound flavin adenine dinucleotide (FAD) as the redox cofactor. The smaller cytochrome domain (residues 1-107) encapsulates four c-type haems which are organised in a dog-leg arrangement. The enzyme catalyses the conversion of fumarate to succinate with a kcat of 509 ± 15 s-1 and Km of 25 ± 2 mM at pH 7.2, 25°C, which corresponds to a catalytic efficiency of 2.1 x 107 M-1s-1. The reverse reaction, succinate oxidation, proceeds less efficiently with a kcat of 0.7 ± 0.02 s-1 and a Km of 0.8 ± 0.1 mM. This results in a catalytic efficiency of 933 M-1 s-1 at pH 8.5. The efficiency of succinate oxidation is a factor of 2 x 104 less than for fumarate reduction confirming that flavocytochrome c3 is a unidirectional fumarate reductase. Flavocytochrome c3 differs in this respect from the wider family of fumarate reductases which are freely reversible. The effect of pH on both fumarate reduction and succinate oxidation has been studied. The results yield a pKa of 7.32 ± 0.5 for fumarate reduction and 8.6 ± 0.1 for succinate oxidation, indicative of the presence of an essential histidine at the active site of the enzyme. A programme of site-directed mutagenesis was undertaken with a view to probing the catalytic mechanism. Kinetic analysis of mutated forms of flavocytochrome c3, in conjunction with the recently determined crystal structure, has allowed the proposal of a mechanism for the reaction. Substitution of the active site histidines (365 and 504) by alanine resulted in dramatic changes in the pH profile for fumarate reduction. The overall rate of reaction was lowered 100-fold in each case. Active-site arginines 402 and 381 were also studied. The substitution of arginine 402 by alanine results in a complete loss of fumarate reductase activity, leading to the assignment of this residue as the essential active site base. Arginine 381, on the other hand, is likely to be involved in a more peripheral role, possibly in stabilising the transition state structure. Aspartate 195 was also studied but the substitution of this to alanine was found to have no effect on the catalytic parameters.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available