Sulfite oxidizing enzymes

Abstract

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

Fig. 1

Selected amino acids and ligands near Mo centers of wild type proteins of chicken SO (left), A. thaliana SO (middle), and S. novella SDH (right). Water molecules are shown in red spheres. Hydrogen bonds are shown in dashed green lines. Coordinates for this figure are from the Brookhaven Protein Data Bank; PDB entries for chicken SO, A. thaliana SO and S. novella SDH are 1SOX, 1OGP, and 2BLF, respectively. The structures of chicken SO R138Q [24] and S. novella SDH Y236F [30] mutants have been recently reported.

Fig. 2

Cα traces of superimposed S. novella SDH (green) and chicken SO (blue) crystal structures with the heme moieties shown in a space filling mode (red and magenta in SO and SDH, respectively), and the Mo cofactor in yellow. The superposition is in an orientation to demonstrate the very different cytochrome interaction sites of the two models. Note that the loop connecting the Mo and heme domains in chicken SO is disordered in the crystal structure, indicating flexibility of this loop as a tether for the heme motion during SO catalysis.

Fig. 3

Proposed oxidation state changes occurring at the Mo and Fe centers of native animal SO during the catalytic oxidation of sulfite and the concomitant reduction of (cyt c)_ox. Only the equatorial oxygen atom among the ligands of Mo is shown for clarity. Note that the intermediate Fe^II/Mo^V–OH in the reductive half reaction is EPR detectable. The one-electron reduction of Fe^III, indicated by a dotted arrow connecting Mo^VIFe^III and Mo^VIFe^II, can be initiated with a laser pulse in a solution containing dRF and the sacrificial electron donor semicarbazide. The subsequent IET between Mo^VIFe^II and Mo^VFe^III, which is of particular interest in this review, is highlighted in red. The rate constants of forward and reverse IET (k_f and k_r, respectively) are defined in the text.

Fig. 4

Proposed chemical mechanism for the reductive half reaction of SO [60].

Fig. 5

Transient obtained at 555 nm upon photoexcitation of a solution containing wild type human SO, dRF, and 0.5 mM fresh semicarbazide hydrochloride (pH 7.4). The portion of the figure outlined by the orange box points to heme reduction by dRFH^•; this process is pseudo first order, and its rate depends on protein concentration. The dark blue box points to heme reoxidation due to the subsequent IET between the Mo and Fe centers; this process is independent of protein concentration, consistent with its intraprotein nature. The red solid line indicates a single-exponential fit to the IET phase. K_eq = b/a.

Fig. 6

Proposed docking of the heme domain to the Mo domain in vertebrate SO that could move the Mo and Fe centers into closer proximity (structure II) than that observed in the crystal structure of dimeric SO (structure I). Subsequently, the transient internal complex (structure II) may facilitate rapid IET to generate structure III. A possible docking site near the Mo domain in the dimeric form is marked by a green arrow.

Fig. 7

Potential pathways for electron transfer from the Fe to Mo centers in S. novella SDH. The red block arrow shows pathways through hydrogen bonds, whereas the green arrow shows pathways through aromatic residues. SorA residues are yellow, SorB residues are blue.