The structure of homogeneous Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) has been determined using 2D NMR methods and molecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907−916) reported several possible structures for CoBLM A2 green compatible with their NMR data acquired on a mixture of CoBLM A2 green and A2 brown forms. The availability of the pure CoBLM A2 green, which is stable for months at neutral pH, has allowed the complete assignments of the 1H and 13C chemical shifts, observation of 55 intramolecular NOEs, and determination of 15 coupling constants allowing the definition of dihedral angles. These results are a prerequisite to determining its structure with duplex DNA of a defined sequence (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). Two screw sense isomers each containing two possible axial ligands (the primary amine of the β-aminoalanine and the carbamoyl nitrogen of the mannose) were considered as viable candidates for the structure of CoBLM A2 green. Using the NMR constraints and molecular dynamics calculations, the structures of all four isomers were generated. One set of screw sense isomers can be readily eliminated from considerations based on violations of NOE and dihedral angle constraints. The other screw sense isomer containing either one or the other of the postulated axial ligands has been examined in some detail. The structure containing the primary amine of β-aminoalanine as the axial ligand is favored on the basis of coupling constants and NOE arguments, potential energy considerations, model studies, and studies with analogs of BLM. The favored structure is compact with the bithiazole moiety folded back underneath the equatorial plane of the metal binding domain, on the same face as the hydroperoxide ligand. The geometry of the peptide linker is very well defined by the observed coupling constants in the valeryl and threonine moieties of the linker. CoBLM A2 green has been studied with two self-complementary oligonucleotides, d(CCAGGCCTGG) and d(CCAGTACTGG). Both of these oligomers possess a single, UV light-mediated cleavage site (C and T, respectively). In addition, fluorescent quenching studies have allowed the determination of the first sequence-specific dissociation constants of 1.7 × 10-7 and 1.5 × 10-7 M, respectively. Titration of CoBLM A2 green with each of these oligomers reveals a 1:1 complex in slow exchange on the NMR time scale. The upfield shifts of the bithiazole protons in both of these complexes are indicative of a partial intercalative mode of binding. The stage is now set for the determination of the structure of the CoBLM A2 green bound sequence specifically to DNA.

The structural similarity of HOO-CoBLM bound to d(GpT) in d(CCAGTACTGG) compared to a d(GpC) motif suggests a general paradigm for the binding of HOO-CoBLM to DNA and, by analogy, for the binding of the biological significant entity HOO-FeBLM.

Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady-state kinetic parameters have been determined: kcat(SO3(2-) = 0.31 mol SO3(2-).s-1.mol heme-1, Km = 0.06 mM; kcat(NO2-) = 0.038 mol NO2-.s-1.mol heme-1, Km = 0.028 mM; kcat(NH2OH) = 29 mol NH2OH.s-1.mol heme-1, Km = 48 mM. A detailed comparison is made with the Escherichia coli and spinach assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.