Lewis M. Siegel

Sulfite reductase structure at 1.6 A: evolution and catalysis for reduction of inorganic anions.

Fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen are catalyzed by sulfite and nitrite reductases. The crystallographic structure of Escherichia coli sulfite reductase hemoprotein (SiRHP), which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia, was solved with multiwavelength anomalous diffraction (MAD) of the native siroheme and Fe 4S4 cluster cofactors, multiple isomorphous replacement, and selenomethionine sequence markers. Twofold symmetry within the 64-kilodalton polypeptide generates a distinctive three-domain α/β fold that controls cofactor assembly and reactivity. Homology regions conserved between the symmetry-related halves of SiRHP and among other sulfite and nitrite reductases revealed key residues for stability and function, and identified a sulfite or nitrite reductase repeat (SNiRR) common to a redox-enzyme superfamily. The saddle-shaped siroheme shares a cysteine thiolate ligand with the Fe4S4 cluster and ligates an unexpected phosphate anion. In the substrate complex, sulfite displaces phosphate and binds to siroheme iron through sulfur. An extensive hydrogen-bonding network of positive side chains, water molecules, and siroheme carboxylates activates S-O bonds for reductive cleavage.

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme† chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.

[46] Siroheme: Methods of Isolation and characterization

Publisher Summary This chapter describes the methods of characterization and isolation related to siroheme. A number of sulfite and nitrite reductase enzymes from bacteria, fungi, and plants are found to contain a new type of heme-related prosthetic group that is known as siroheme. Certain of these enzymes are associated in vivo with phosphorylating respiratory chains and are therefore presumably normally membrane bound. Most of the enzymes studied, however, serve a purely biosynthesic role in the assimilation of sulfate and nitrate; there is no evidence for membrane association of these enzymes. Although siroheme enzymes generally yield siroheme itself upon extraction with acetone/HCl, the desulfoviridin-type of sulfite reductase from Desulfovibrio gigas and D. vulgaris yields sirohydrochlorin under such conditions. The properties of sirohydrochlorin compared with those of siroheme in media used during the extraction and purification of the latter compound from enzyme systems are described by Murphy and Siegel.

Flavin interaction in NADPH-sulfite reductase.

E. coli NADPH-sulfite reductase, depleted of FMN but retaining its FAD, has been prepared by photoirradiation of native enzyme in 30% — saturated ammonium sulfate. FMN-depleted enzyme loses its ability to reduce (using NADPH) ferricyanide, cytochrome c, sulfite, or the enzyme’s own heme-like chromophore. However, the FAD remains rapidly reducible by NADPH, and the FMN-depleted enzyme retains NADPH-acetylpyridine NADP* transhydrogenase activity. Thus, FAD can serve as entry port for NADPH electrons, and FMN is required for further transmission along the enzyme’s electron transport chain. These data, plus other studies, have enabled us to suggest a mechanism for catalysis which involves FAD cycling between the fully-oxidized and fully-reduced forms while FMN cycles between fully-reduced and semiquinone. This mechanism, which includes a disproportionation step, permits a “step-down” from the twoelectron donor, NADPH, to a succession of equipotential one-electron transfer steps.

Optical and electron paramagnetic resonance spectroscopic studies on purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans contains a molybdenum cofactor very similar to that found in a number of other molybdenum-containing hydroxylases. (A. nidulans contains two purine hydroxylases, I and II, related to each other by possession of a common cofactor and overlapping substrate specificity.) Addition of reducing substrates effects bleaching of the visible absorption spectrum of the enzyme, the decrease in absorbance at 450 nm being linearly proportional to that at 550 nm. No increase in absorption at longer wavelengths was observed during such titrations. Electron paramagnetic resonance studies of reduced samples of native and modified enzyme species showed the presence of a number of Mo(V) signals (gav = 1.97), exhibiting H hyperfine coupling, comparable to those in the corresponding enzymes from other sources. The enzyme possesses two non-heme-iron-sulfur centers, one (Fe2S2)I with gav less than 2.0 and the other (Fe2S2)II with gav greater than 2.0. The flavin radical signal observed at pH 7.8 had a linewidth of 1.5 mT, indicating it to be the anionic form FAD- . In this respect purine hydroxylase II is unique among all molybdenum-containing hydroxylases studied to date.

[209] TPNH-sulfite reductase (Escherichia coli)☆☆☆★

Publisher Summary This chapter discusses the methods of preparation of TPNH-Sulfite Reductase ( Escherichia coli ). Sulfite reductase is a key enzyme on the regulated pathway of cysteine biosynthesis in the enterobacteria, and the level of enzymatic activity is closely responsive to the intracellular cysteine content. It is, therefore, possible to derepress enterobacteria ior sulfite reductase by growth on limiting sulfur sources, so that under favorable conditions the enzyme can comprise approximately 0.5% of the soluble cell protein. Sulfite reductases from enterobacteria and yeast are high molecular weight iron-flavoproteins that can utilize either TPNH or artificial dyes (such as reduced methyl viologen) as donors for the six-electron reduction of sulfite to sulfide. A unit of activity is defined as that amount of enzyme that catalyzes the oxidation of 1 micromole of TPNH per minute with sulfite as electron acceptor under the above assay conditions. The purified enzyme appears homogeneous by sedimentation analysis in the Beckman Model E ultracentrifuge and by electrophoresis in both polyacrylamide gel (pH 9) and cellulose acetate strip (pH 7.7) supporting media.

Paramagnetic Proton NMR Methods Used in Studying the Hemeprotein Subunit of Escherichia coli Sulfite Reductase

Publisher Summary This chapter discusses paramagnetic proton nuclear magnetic resonance (NMR) methods used in studying the hemeprotein subunit of Escherichia coli sulfite reductase. NMR spectroscopy of solutions containing paramagnetic metalloproteins is a rapidly developing, specialized area in the larger field of protein NMR. In the presence of a paramagnetic prosthetic group, hyperfine-shifted resonances may be observed outside the normal diamagnetic spectral envelope, and can often be used as a probe of the proteins electronic and molecular structure near the active site. The chapter presents some of the principles and techniques necessary to successfully utilize paramagnetic NMR spectroscopy. Structural information about the active site can be obtained from the NMR spectra of paramagnetic metalloproteins, particularly in the case of coupled metal systems. Resonance assignments can be achieved by a variety of techniques, including isotopic substitution, ID-NOE measurements, 2D NOESY/COSY maps, variable temperature intercepts, differential dipolar relaxation, and comparison with model compounds. The size and magnitude of hyperfine-shifted resonances along with their temperature dependence and relaxation properties are also valuable in understanding the electronic and magnetic properties of paramagnetic proteins.

Alkaline low spin form of sulfite reductase hemeprotein subunit

The reversible reduction and reoxidation of Escherichia coli sulfite reductase hemeprotein subunit at pH 9.9 produces high and low spin ferric species, the latter with properties distinct from any alkaline low spin yet reported. With virtually no effect on the 298 degrees K optical spectrum, chloride drastically reduces the low spin EPR intensity and produces a high spin conformer pattern like that seen at pH 11. The distribution of g = 5 and g = 2.29 species in the doubly-reduced enzyme is also pH-sensitive.

A novel system involving exchange-coupling between a heme and an ironsulfur cluster

Abstract The β-subunits of E. coli sulfite reductase (SiR) contain an Fe-isobacteriochlorin, termed siroheme, and a [4Fe-4S] cluster. Mossbauer and EPR studies [1] of oxidized SiR have demonstrated that the siroheme and the ironsulfur cluster are exchange coupled. Such a coupling implies a covalent link between the two chromophores; it is reasonable to assume that the attached to the heme iron by an as yet unspecified bridging ligand. In oxidized SiR the iron-sulfur cluster is in the 2+ oxidation state, a state in which [4Fe-4S] clusters are normally diamagnetic. Through exchange interactions with the siroheme the cluster acquires paramagnetism; the experimental observations have been explained qualitatively in a model which involves isotropic exchange between the heme iron and one site of the cluster [2]. Recent studies [3] of one-electron and two-electron reduced SiR, a nitrate ‘turnover’ (ferroheme-NO) complex, and studies of SiR in chaotropic agents that the coupling is maintained in may oxidation-, spin-, and complexation-states of the enzyme. We have also studied SiR complexed to cyanide (in three oxidation states) and carbon monoxide. Exchange coupling is indicated in the oxidized cyano complex; in the reduced CN − and CO complexes the heme is low-spin ferrous and thus in a state unfavorable for the development of interatomic exchange.