Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase

There is now overwhelming evidence supporting a common mechanism for fumara te reduction in the respiratory fumarate reductases. The X-ray structures o f substrate-bound forms of these enzymes indicate that the substrate is wel l positioned to accept a hydride from FAD and a proton from an arginine sid e 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] h as strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic an alyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme . The crystal structure of the R402A mutant (2.0 Angstrom 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 en zyme, the R402A mutant is inactive under all the conditions that were studi ed. This implies that a water molecule, in this position in the active site , cannot function as the proton donor for fumarate reduction. In contrast t o the R402A mutation, both the R402K and R402Y mutant enzymes are active. A lthough this activity was at a very low level (at pH 7.2 some 10(4)-fold lo wer than that for the wild type), it does imply that both lysine and tyrosi ne 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 resolu tion) show that distances from the lysine and tyrosine side chains to the n earest carbon atom of fumarate are similar to3.5 Angstrom, clearly permitti ng 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 transf er from FAD to fumarate) and a Bronsted acid (donating the proton to the su bstrate to complete the formation of succinate).