Spectroscopic Characterization of YedY: The Role of Sulfur Coordination in a Mo(V) Sulfite Oxidase Family Enzyme Form

Abstract

Electronic paramagnetic resonance, electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, A. thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from S. novella. Low-energy dithiolene→Mo and cysteine thiolate→Mo charge transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. Namely, a more acute Ooxo-Mo-SCys-C dihedral angle results in increased cysteine thiolate S→Mo charge transfer and a high g1 in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

“…The disappearance of the absorbance centered at 503 nm is consistent with our assignment of the positive potential redox process being Mo-based; dithiolene-S ligand to metal charge transfer processes give rise to this spectral feature so it should be a reporter signal for changes to the metal redox state (18). Both dcV trumpet plot data and low-intensity FTacV harmonic currents indicate that the Mo(V/IV) redox reaction has a slow electron transfer rate, from 3 to 6 s −1 , which suggests structural reorganization.…”

The Mononuclear Molybdenum Enzymes

“…The highly axial g tensor has been attributed to the extremely covalent nature of the metal–sulfur ligation (from the enedithiolate as well as the cysteine). 364 Electronic absorption transitions at 11 900 cm −1 (840 nm), 19 122 cm −1 (523 nm), and 21 578 cm −1 (463 nm) have been assigned to the following charge-transfer interactions, respectively: out-of-plane enedithiolate S(p) → Mo(d x 2 −y 2), out-of-plane Cys S(p) → Mo(d x 2− y 2 ), and out-of-plane Mo(d x 2− y 2 ) + Cys S(p) → Mo(d xz ) + Mo(d yz ); all three transitions have very weak oscillator strengths below 0.01 (and ɛ < 1000 M −1 cm −1 ). These assignments may well hold for all members of the sulfite oxidase family, at least for those Mo(V) forms whose EPR do not manifest hyperfine coupling to protons, although the energies of the transitions can be expected to vary.…”

The molybdenum oxotransferases and related enzymes

“…73 The exceptionally high axial nature of the g tensor has been attributed to the extremely covalent nature of the metal-sulfur ligation (from the enedithiolate as well as the cysteine). 73 Electronic absorption transitions at 11 900 cm −1 (840 nm), 19 122 cm −1 (523 nm) and 21 578 cm −1 (463 nm) have been assigned to the following charge-transfer interactions, respectively: out-of-plane enedithiolate S( p) → Mo(d x 2 −y 2), out-of-plane Cys S( p) → Mo(d x 2 −y 2) and out-of-plane Mo(d x 2 −y 2) + Cys S( p) → Mo(d xz ) + Mo(d yz ). 75 All three transitions have very weak oscillator strengths, below 0.01 (and ε < 1000 M −1 cm −1 ).…”