CRYSTALLOGRAPHIC STUDIES OF THE MULTI-ELECTRON REDUCTIONS CATALYZED BY THE SIROHEME AND IRON-SULFUR-CLUSTER CONTAINING ENZYME SULFITE REDUCTASE. Brian R. Crane, Lewis M. Siegel and Elizabeth D. Getzoff, Department of Molecular Biology, The Scripps Research Institiute, La Jolla California, 92037, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Reductions of the inorganic substrates sulfite and nitrite are important for the assimilation of sulfur and nitrogen into the biosphere and for the dissimilation of oxidized forms of these elements during anaerobic energy procurement.

To further understand the enzymatic redox chemistry involved in these processes we have characterized high-resolution crystallographic structures of the 64 kD E.coli NADPH sulfite reductase hemoprotein (SiRHP) in different oxidation states, and in complex with inhibitors, substrates, reaction intermediates and products.

When supplied with suitable electron donors, SiRHP catalyzes the six-electron reduction of sulfite to sulfide, and nitrite to ammonia, without releasing any detectable intermediates. The protein's internal twofold symmetry relates gene-duplicated modules that have diverged to bind different cofactors for assembly of the active center. SiRHP's unique active center consists of a siroheme, (an unusual iron-tetrahydroporphyrin of the isobacteriochlorin family), coupled structurally and electronically to an [4Fe-4S] cluster via a cysteinate sulfur bridge.

The ligand binding site, on the siroheme's distal face, has been optimized for electrophilic catalysis, and has been constructed to provide preferred locations for recognizing the functional groups of ligands with varied size, shape and charge.

Complex interplays relate the electronic states of the prosthetic groups, the interactions of ligands with the cofactors and the protein, and the structural coupling of the cofactors to the protein moiety. Correlated changes occuring in the electronic state of the siroheme iron and the conformations of active-site residues and exposed loops sequester and activate the substate sulfite for reduction.