Elucidating the catalytic mechanism of sulfite oxidizing enzymes using structural, spectroscopic, and kinetic analyses

Abstract

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification and in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates, the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid kinetic studies of PSO are also discussed.

Figure 1

Top row: Ribbon diagrams of the crystal structures of SOEs: 1A CSO (26), 1B PSO (32), 1C SDH (29); blue represents the molybdenum and C-terminal domain; red represents the heme domain. Bottom row: Selected amino acid residues near the molybdenum active site of the corresponding SOEs of the top row.

Figure 2

Sequence alignment of the flexible tether regions of CSO and HSO. Amino acids highlighted in red are conserved between the two species, while those in blue are similar. Mutations discussed in this work are indicated in the HSO sequence: proline residues mutated to alanines are in bold type, and deleted residues are underlined.

Figure 3

Top: superposition of the active site structures of SDH^wt and SDH^R55M, showing that the M55 side chain (red) in SDH^R55M does not occupy the same space as R55 (blue) in SDH^wt. M55 bends away, packing into a small cavity between the side chains of L121 and Q33, and the space previously occupied by R55 in SDH^wt is empty (31). Bottom: structure of the active site of SDH^H57A, showing the two disordered conformations of the R55 side chain (31).

Figure 4

Proposed oxidation state changes occurring at the Mo and Fe centers of animal SO during the catalytic oxidation of sulfite and the concomitant reduction of (cyt c)_ox.

Figure 5

Proposed structures of the molybdenum active sites of SOEs from pulsed EPR experiments and DFT calculations for the lpH (50), blocked (61), and hpH forms (58). The blocked form shown on the left has bound reactant (sulfite); the form shown on the right has bound product (sulfate). See text and (61) for details.

Figure 6

Kinetic 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 the initial 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. Copyright Elsevier publishing; reproduced from Feng, C., Tollin, G., and Enemark, J. H. (2007) Sulfite Oxidizing Enzymes, Biochim. Biophys. Acta 1774, 527-539, with permission.

Figure 7

IET Rate Constants for the Proline to Alanine Tether Mutants (79).

Figure 8

IET Rate Constants for Tether Deletion Mutants (79).

Figure 9

Spectroelectrochemical titration of the b₅ heme of wild-type HSO at pH 7.5 and 27°C (79). The inset shows the fit of the data to eq. 11 at 413 nm (78); black = wt; red = ΔKVATV mutation.

Scheme 1

Possible Reaction Pathways for the Catalytic Oxidation of Sulfite by SDH or SO^{a a}For the sake of simplicity, the enzyme and substrate are depicted as Mo^VIO and SO₃^2-, respectively. The pathways differ in the sequence of steps which transform the enzyme-substrate complex, Mo^IV(OSO₃^2-), into product (sulfate) and the reoxidized Mo(VI) state of the enzyme. The pathway colored red is the one commonly proposed, in which product release precedes reoxidation of the enzyme. For the pathway colored black, the enzyme-substate complex is oxidized by two electrons prior to product release (34). See the text for additional discussion.

Scheme 2

Overview of the current state of research on sulfite oxidizing enzymes (SOEs). From bioinformatic analyses, the diverse sulfite oxidase family of proteins that contains the same molybdenum cofactor center (1) can be classified into at least three groups (63, 85). This review has focused on the SOEs. Animal sulfite oxidases possess two redox active domains and present fundamental biophysical problems relating to intramolecular electron transfer (IET), the relationship of IET rates to steady-state kinetics, the overall conformation of the protein, and the molecular dynamics of the motion of the two domains relative to one another. The effects of extensive mutations of the tether connecting the heme and molybdenum domains of human SO have been discussed here. However, as yet there are no X-ray structural results of the intact protein for any of these variants. The detailed structures of the molybdenum centers of these variants have been determined from analysis of high resolution pulsed EPR spectra of their Mo(V) states as a function of pH, anions in the media, and mutations of nearby amino acid residues. A long term hope is that these studies will contribute to the continuing development of molecular medicine to treat sulfite oxidase deficiency (13, 16).