Spectroscopic and Kinetic Studies of Y114F and W116F Mutants of Me2SO Reductase from Rhodobacter capsulatus

Abstract

Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me 2 SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me 2 SO to reduced enzyme, the result of lower stability of the E red ⅐Me 2 SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wildtype enzyme, consistent with Tyr-114 interacting with bound Me 2 SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second MoAO group. Reduction of the mutant with sodium dithionite and reoxidation with Me 2 SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This "redoxcycled" mutant exhibits a Me 2 SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover.Dimethyl sulfoxide reductase catalyzes the reduction of dimethyl sulfoxide (Me 2 SO) to dimethyl sulfide (DMS) 3 and is a member of a large class of mononuclear molybdenum-containing enzymes (1-3) having an L 2 MoO(X) core (where L represents a pyranopterin cofactor coordinated to the molybdenum via an enedithiolate side chain, and X represents a ligand in most cases provided by the polypeptide, a serinate in the case of Me 2 SO reductase). Two forms of Me 2 SO reductase exist, as exemplified by the enzymes from Escherichia coli (a heterotrimer consisting of the DmsABC gene products) and Rhodobacter species (the monomeric DorA protein). Both are components of anaerobic respiratory chains and catalyze the reductive abstraction of oxygen from Me 2 SO according to the following stoichiometry using electrons derived from the respiratory pool.The Rhodobacter sphaeroides DorA enzyme is a soluble periplasmic protein of 85 kDa possessing the molybdenum cofactor as the sole redox-active center. Because of the distinctive absorption features of its molybdenum center, the DorA enzyme has become a paradigm for understanding the electronic structure of molybdenum centers of the L 2 MoO(X) variety (4 -12). In addition to steady-state kinetics under a variety of conditions, rapid reaction studies with both the Rhodobacter capsulatus (10) and R. sphaeroides enzyme (13) have also been reported. With the R. sphaeroides enzyme, the reaction of reduced enzyme with Me 2 SO at low pH is biphasic. A fast [Me 2 SO]-dependent phase (k lim ϳ 1000 s Ϫ1 ) yields a spectrally distinct E red ⅐Me 2 SO complex, with absorption maxima at 490 and 550 nm, followed by a slower oxygen abstraction step (35 s Ϫ1 ) that yields DMS and E ox (13). The hyperbolic dependence of the rapid phase on [Me 2 SO] gives a K d Me 2SO of ϳ155 M and indicates tha...

Unexpected Reaction Promoted by NH+···O=Mo Hydrogen Bonds in Nonpolar Solvents

“…[40,41] Given that the iso-lated form of the W116F mutant of DMSOR from Rhodobacter capsulatus showed a five-coordinate structure, accompanied by dissociation of the Q pterin from the molybdenum, the hydrogen bond involving the tryptophan residue has been considered to stabilise the Mo-S bond, [42] whereas the Mo-S bond is recovered in the catalytic cycle. [6] Stabilisation by hydrogen bonding is reasonably explained in the present study. Such interactions should be stabilised in the hydrophobic environment around the active site of the enzyme [2] as well as in our model complex.…”

“…[4,[7][8][9] In the active site, the conserved tryptophan (Trp) residue forms an NH···O=Mo hydrogen bond, which stabilises the coordination of the Q pterin to the Mo VI centre, but does not significantly affect the enzymatic activity. [6] Similar hydrogen bonds involving a histidine residue have been reported for the members of the DMSOR family. [10,11] A number of models of the DMSOR family have been reported, [12] and model complexes containing ligands closely related to MPT have recently been synthesised.…”

“…Benzenedithiol containing acylamino substituents at positions 3 and 6, 3,6-[(4-tBuC 6 Interestingly, a solution of (LuH) 2 [1] was unaltered in a slightly polar solvent such as chloroform.…”

The Mononuclear Molybdenum Enzymes

“…Mutagenesis studies of Tyr 114 385 and Trp 116 385b have probed the catalytic roles of these residues. Trp 116 is within hydrogen-bonding distance of the Mo=O of oxidized enzyme, and Tyr 114 (which is a valine in the otherwise closely related TMAO reductase from Shewanella massilia , see below) has been proposed to hydrogen bond to the oxygen of DMSO in the course of its reaction with reduced enzyme.…”

Comparison of the Active-Site Design of Molybdenum Oxo-Transfer Enzymes by Quantum Mechanical Calculations

“…The calculated barrier is somewhat too large, compared to experiments, 62 kJ/mol. 10,69 The reason for this is partly the DFT functional, partly the omission of the surrounding enzyme. Calibration calculations with the local CCSD(T0) method have shown that energies calculated with the current methodology have errors of 19-45 kJ/mol for the reaction mechanism of DMSOR.…”