Molecular Basis of Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes Is Revealed by High Resolution Structure of a Heterodimeric Complex of the Catalytic Molybdopterin Subunit and a c-Type Cytochrome Subunit

Abstract

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 Å) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit. Sulfite oxidases (SO(s))1 and nitrate reductases are members of a subclass of molybdopterin enzymes characterized by the presence of a MoO 2 metal center. The enzymes typically contain a molybdopterin cofactor (Moco) implicated in substrate binding and oxidation and a heme cofactor for molybdenum reoxidation. Three types of sulfite-oxidizing enzymes have been characterized so far, namely the homodimeric, heme b and molybdenum-containing enzymes from higher animals and birds (1, 2), the homodimeric, molybdenum-containing enzymes from plants (3) and a heterodimeric, heme c and molybdenum-containing bacterial enzyme (4) that cannot transfer electrons to molecular oxygen and is therefore classified as a sulfite dehydrogenase. Of these enzymes, the sulfite oxidases from higher animals and birds have been studied in great detail, as in humans a deficiency in SO activity is linked to severe developmental retardation and early death of the affected individuals (2, 5). The crystal structures of two homodimeric eukaryotic sulfite oxidases, CSO from chicken liver (2) and PSO from Arabidopsis thaliana (6), have been determined and have provided structural details of the molybdenum center and substrate binding pocket. During catalysis two electrons are transferred from the substrate, sulfite, to the fully oxidized Mo(VI) form of the active site, which is then reduced to the Mo(IV) state. The two electrons are then transferred sequentially from the molybdenum to an electron acceptor (PSO) or to the heme cofactor, which can be present either as a domain (e.g. CSO, human sulfite oxidase (HSO)) or subunit (e.g. bacterial sulfite dehydrogenase) of the enzyme. Using the structural information and the results from site-directed mutagenesis studies on HSO (7-9) it...

Structural Studies of the Molybdenum Center of the Pathogenic R160Q Mutant of Human Sulfite Oxidase by Pulsed EPR Spectroscopy and 17O and 33S Labeling

“…Two possibilities for the trans ligand X (O or N) were discussed recently from EXAFS and DFT studies on R160Q SO. 13 From our studies, only X = O remains plausible because no 14 N modulations, which are easily detectable by ESEEM, were observed.…”

Direct Demonstration of the Presence of Coordinated Sulfate in the Reaction Pathway of Arabidopsis thaliana Sulfite Oxidase Using 33S Labeling and ESEEM Spectroscopy

“…Note that the largest value of α is only 15° (reached for η = 1), and therefore s α < 0.26. The parameter C η : (3) is introduced because it will be encountered in numerous expressions below. Since 0 ≤ η ≤ 1, C η ≈ 1 with an accuracy of ~ 15% (for η = 1) or better.…”

Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

“…One candidate is Tyr, 343 which has been implicated as being important in catalytic turnover of the enzyme 43 and in electron transfer between the cytochrome b5 moiety and the Moco. 44 Furthermore, the bacterial sulfite dehydrogense of Starkeya novella 45,46 48 Free tyrosine in aqueous solution has a side-chain pKa of 10.1, but this will be significantly shifted (to a lower value) by the presence of the highly basic residues in the active site of SO, so that one of the two observed pKa values of R160Q SO could be that of Tyr. 343 The two low-pH species are quite similar EPR signals (different g-values, but similar g-anisotropies, a similar lack of resolved proton hyperfine splitting, and indistinguishable redox potentials), and it seems likely that the two low-pH species are structurally similar.…”