An MCD spectroscopic study of the molybdenum active site in sulfite oxidase: insight into the role of coordinated cysteine.

Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below approximately 17000 cm(-1). This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d(xy) orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo-Scys sigma-bond) and Mo d(xy) orbitals. The spectroscopically determined cysteine sulfur p-Mo d(xy) bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O-Mo-Scys-C dihedral angle of approximately 90 degrees and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d(xy), effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22250 cm(-1) positive C-term feature in the MCD as the cysteine S(sigma)-->Mo d(xy) charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.

Control of oxo-molybdenum reduction and ionization potentials by dithiolate donors.

The compounds (L-N3)MoO(qdt) and (L-N3)MoO(tdt) [(L-N3) = hydrotris(3,5-dimethyl-1-pyrazolyl)borate; tdt = toluene-3,4-dithiolate; qdt = quinoxaline-2,3-dithiolate] have been studied by cyclic voltammetry and photoelectron, magnetic circular dichroism, and electronic absorption spectroscopies, and the experimental data have been interpreted in the context of ab initio molecular orbital calculations on a variety of dithiolate dianion ligands. The PES data reveal very substantial differences between (L-N3)MoO(qdt) and (L-N3)MoO(tdt) in that the first ionization (originating from the Mo dxy orbital) for (L-N3)MoO(qdt) is about 0.8 eV to deeper binding energy than that of (L-N3)MoO(tdt). This stabilizing effect is also reflected in the solution reduction potentials, where (L-N3)MoO(qdt) is approximately 220 mV easier to reduce than (L-N3)MoO(tdt). A direct correlation between the relative donating ability of a given dithiolate ligand and the reduction potential of the (L-N3)MoO(dithiolate) complex has been observed, and a linear relationship exists between the calculated Mulliken charge on the S atoms of the dithiolate dianion and the Mo reduction potential. The study confirms previously communicated work (Helton, M. E.; Kirk, M. L. Inorg. Chem. 1999, 38, 4384-4385) that suggests that anisotropic covalency contributions involving only the out-of-plane S orbitals of the coordinated dithiolate control the Mo reduction potential by modulating the effective nuclear charge of the metal, and this has direct relevance to understanding the mechanism of ferricyanide inhibition in sulfite oxidase. Furthermore, these results indicate that partially oxidized pyranopterins may play a role in facilitating electron and/or atom transfer in certain pyranopterin tungsten enzymes which catalyze formal oxygen atom transfer reactions at considerably lower potentials.