Identification of the Active Site Acid/Base Catalyst in a Bacterial Fumarate Reductase:  A Kinetic and Crystallographic Study

Abstract

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.

A Threonine on the Active Site Loop Controls Transition State Formation in Escherichia coli Respiratory Complex II

“…The substitution of the active site threonine (FrdA Thr-244/SdhA Thr-254) to alanine caused a much more dramatic effect on complex II enzyme activity compared with the single substitutions of the two histidine residues found at the active site in the soluble Fcc 3 enzyme (26). The results presented here are consistent with act-T being essential for positioning the C1 carboxyl of fumarate and efficient catalysis in the complex II family of enzymes.…”

“…In the complex II enzyme family, hydride transfer occurs with orbital overlap between the flavin isoalloxazine N5 and the C2 of the substrate (2,12,26,27). Charge transfer (CT) complexes with flavins act as intermediates in enzyme catalysis (46).…”

“…Furthermore, in the Fcc 3 enzyme, x-ray crystallography of a mutant enzyme equivalent to E. coli QFR FrdA H232A only showed subtle changes in the position of fumarate; however, the C1 carboxyl group is found in the same twisted conformation as in wild type enzyme (26), indicating that this histidine does not participate in transition state formation. The substitution of the active site threonine (FrdA Thr-244/SdhA Thr-254) to alanine caused a much more dramatic effect on complex II enzyme activity compared with the single substitutions of the two histidine residues found at the active site in the soluble Fcc 3 enzyme (26).…”

“…This suggests that the removal of the act-T O␥ hydrogen bond affects both substrate binding and transition state stabilization. In contrast, substitutions of other active site residues equivalent to E. coli FrdA His-232 and His-354 were mainly shown to affect substrate binding (12,26,51).…”

3-Nitropropionic Acid Is a Suicide Inhibitor of Mitochondrial Respiration That, upon Oxidation by Complex II, Forms a Covalent Adduct with a Catalytic Base Arginine in the Active Site of the Enzyme

“…The residue equivalent to Arg 297 is believed from structural studies and site-directed mutagenesis to serve as a catalytic acid in the soluble FCc FRD (38,39), donating a proton to one end of the double bond while a hydride is transferred from flavin to the other end. Assuming that succinate oxidation in SQR occurs by the reverse of this mechanism, Arg 297 should act as a general base catalyst to abstract a proton from one of the central carbons, whereas a hydride is transferred from the other to the flavin.…”