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 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 Brønsted acid (donating the proton to the substrate to complete the formation of succinate).

Catalysis in fumarate reductase.

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study.

The active sites of respiratory fumarate reductases are highly conserved, indicating a common mechanism of action involving hydride and proton transfer. Evidence from the X-ray structures of substrate-bound fumarate reductases, including that for the enzyme from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], indicates that the substrate is well positioned to accept a hydride from N5 of the FAD. However, the identity of the proton donor has been the subject of recent debate and has been variously proposed to be (using numbering for the S. frigidimarina enzyme) His365, His504, and Arg402. We have used site-directed mutagenesis to examine the roles of these residues in the S. frigidimarina enzyme. The H365A and H504A mutant enzymes exhibited lower k(cat) values than the wild-type enzyme but only by factors of 3-15, depending on pH. This, coupled with the increase in K(m) observed for these enzymes, indicates that His365 and His504 are involved in Michaelis complex formation and are not essential catalytic residues. In fact, examination of the crystal structure of S. frigidimarina fumarate reductase has led to the proposal that Arg402 is the only plausible active site acid. Consistent with this proposal, we report that the R402A mutant enzyme has no detectable fumarate reductase activity. The crystal structure of the H365A mutant enzyme shows that, in addition to the replacement at position 365, there have been some adjustments in the positions of active site residues. In particular, the observed change in the orientation of the Arg402 side chain could account for the decrease in k(cat) seen with the H365A enzyme. These results demonstrate that an active site arginine and not a histidine residue is the proton donor for fumarate reduction.