A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Brønsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Brønsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Brønsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.

Role of His505 in the soluble fumarate reductase from Shewanella frigidimarina.

The X-ray structure of the soluble fumarate reductase 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] clearly shows the presence of an internally bound sodium ion. This sodium ion is coordinated by one solvent water molecule (Wat912) and five backbone carbonyl oxygens from Thr506, Met507, Gly508, Glu534, and Thr536 in what is best described as octahedral geometry (despite the rather long distance from the sodium ion to the backbone oxygen of Met507 (3.1 A)). The water ligand (Wat912) is, in turn, hydrogen bonded to the imidazole ring of His505. This histidine residue is adjacent to His504, a key active-site residue thought to be responsible for the observed pK(a) of the enzyme. Thus, it is possible that His505 may be important in both maintaining the sodium site and in influencing the active site. Here we describe the crystallographic and kinetic characterization of the H505A and H505Y mutant forms of the Shewanella fumarate reductase. The crystal structures of both mutant forms of the enzyme have been solved to 1.8 and 2.0 A resolution, respectively. Both show the presence of the sodium ion in the equivalent position to that found in the wild-type enzyme. The structure of the H505A mutant shows the presence of two water molecules in place of the His505 side-chain which form part of a hydrogen-bonding network with Wat48, a ligand to the sodium ion. The structure of the H505Y mutant shows the hydroxyl group of the tyrosine side-chain hydrogen-bonding to a water molecule which is also a ligand to the sodium ion. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the mutant enzymes are catalytically active but show markedly different pH profiles compared to the wild-type enzyme. At high pH (above 8.5), the wild type and mutant enzymes have very similar activities. However, at low pH (6.0), the H505A mutant enzyme is some 20-fold less active than wild-type. The combined crystallographic and kinetic results suggest that His505 is not essential for sodium binding but does affect catalytic activity perhaps by influencing the pK(a) of the adjacent His504.