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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 A) 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.
Highlights
Sulfite oxidases (SO(s))1 and nitrate reductases are members of a subclass of molybdopterin enzymes characterized by the presence of a MoO2 metal center
Intramolecular electron transfer (IET) between molybdopterin cofactor (Moco) and the heme is fundamental to the function of the mammalian and bacterial enzymes; the electron transfer pathway between the molybdenum and the heme cofactor has not yet been determined
The subunit structure of the heterodimeric SorAB contrasts with the homodimeric structure of the eukaryotic SOs, enzymatic characterization has established that the enzymes exhibit similar catalytic parameters [4]
Summary
Purification and Crystallization—Recombinant sulfite dehydrogenase was purified and crystallized as described previously [12]. The best molecular replacement solution, determined using the program MOLREP [15, 16], was obtained using the Moco domain of CSO (residues 106 –308) modified by truncation of protein side chains to the atoms common between the two proteins. Several low occupancy heavy atom sites in the derivatives could be determined from the positions of consistent peaks in heavy atom difference and anomalous difference Fourier maps calculated using the molecular replacement phases. Addition of either oxidant (ferricyanide, data not shown) or sulfite to the protein during crystallization appeared to lead to a damaged active site with loss of molybdenum. Subsequent structures of cytochrome-oxidized enzyme and crystals reduced with sulfite for 1–2 min before cryocooling avoided these problems and yielded structures with fully occupied molybdenum sites. Coordinates—Coordinates and structure factors have been deposited in the Protein Data Bank (accession code 2blf and 2bpb)
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