1jry
Crystal structure of Arg402Lys mutant flavocytochrome c3 from Shewanella frigidimarina
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OverviewOverview
There is now overwhelming evidence supporting a common mechanism for, fumarate reduction in the respiratory fumarate reductases. The X-ray, structures of substrate-bound forms of these enzymes indicate that the, substrate is well positioned to accept a hydride from FAD and a proton, from an arginine side chain. Recent work on the enzyme from Shewanella, frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000), Biochemistry 39, 10695-10701] has strengthened the assignment of an, arginine (Arg402) as the proton donor in fumarate reduction. Here we, describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of, the R402A mutant (2.0 A resolution) shows it to be virtually identical to, the wild-type enzyme, apart from the fact that a water molecule occupies, the position previously taken by part of the guanidine group of R402., Although structurally similar to the wild-type enzyme, the R402A mutant is, inactive under all the conditions that were studied. This implies that a, water molecule, in this position in the active site, cannot function as, the proton donor for fumarate reduction. In contrast to the R402A, mutation, both the R402K and R402Y mutant enzymes are active. Although, this activity was at a very low level (at pH 7.2 some 10(4)-fold lower, than that for the wild type), it does imply that both lysine and tyrosine, can fulfill the role of an active site proton donor, albeit very poorly., The crystal structures of the R402K and R402Y mutant enzymes (2.0 A, resolution) show that distances from the lysine and tyrosine side chains, to the nearest carbon atom of fumarate are approximately 3.5 A, clearly, permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that, R402 acts as both a Lewis acid (stabilizing the build-up of negative, charge upon hydride transfer from FAD to fumarate) and a Bronsted acid, (donating the proton to the substrate to complete the formation of, succinate).
About this StructureAbout this Structure
1JRY is a Single protein structure of sequence from Shewanella frigidimarina with NA, HEM, FAD and FUM as ligands. Active as Succinate dehydrogenase, with EC number 1.3.99.1 Full crystallographic information is available from OCA.
ReferenceReference
Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase., Mowat CG, Moysey R, Miles CS, Leys D, Doherty MK, Taylor P, Walkinshaw MD, Reid GA, Chapman SK, Biochemistry. 2001 Oct 16;40(41):12292-8. PMID:11591148
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