Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor, and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9FY62F- and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and x-ray absorption spectroscopies, and by single crystal x-ray diffraction at ~ 1.9 Å resolution in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examined by kinetic studies employing pulse radiolytic generation of O2− and by redox titrations. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidation of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme shows saturation behavior that is not observed in WT-NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl− or Br− and is located close to, but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Y9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies are employed to adjust the redox potential and supply of protons, NiSOD has evolved a similar strategy to control the access of anions to the active site.

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA, and CopA, a copper effluxing P1B-type ATPase. We show here that CupA is a novel cell membrane-anchored Cu(I) chaperone, and that a Cu(I)-binding competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA (sCupA) and the N-terminal metal binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds each harboring a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on sCupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

RcnR (resistance to cobalt and nickel regulator) is a 40-kDa homotetrameric protein and metalloregulator that controls the transcription of the Co(II) and Ni(II) exporter, RcnAB, by binding to DNA as an apoprotein and releasing DNA in response to specifically binding Co(II) and Ni(II) ions. Using X-ray absorption spectroscopy (XAS) to examine the structure of metals bound and lacZ reporter assays of the transcription of RcnA in response to metal binding, in WT and mutant proteins, the roles of coordination number, ligand selection, and residues in the N-terminus of the protein were examined as determinants in metal ion recognition. The studies show that the cognate metal ions, Co(II) and Ni(II), which bind in (N/O) 5 S six-coordinate sites, are distinguished from non-cognate metal ions (Cu(I) and Zn(II)), which bind only three protein ligands and one anion from the buffer, by coordination number and ligand selection. Using mutations of residues near the N-terminus, the N-terminal amine is shown to be a ligand of the cognate metal ions that is missing in the complexes with non-cognate metal ions. The side chain of His3 is also shown to play an important role in distinguishing metal ions. The imidazole group is shown to be a ligand in the Co(II) RcnR complex, but not in the Zn(II) complex. Further, His3 does not appear to bind to Ni(II), providing a structural basis for the differential regulation of RcnAB by the two cognate ions. The Zn(II) complexes change coordination number in response to the residue in position three. In H3C-RcnR, the Zn(II) complex is fivecoordinate, and in H3E-RcnR the Zn(II) ion is bound to six protein ligands. The metric parameters of this unusual Zn(II) structure resemble those of the WT-Ni(II) complex, and the mutant protein is able to regulate expression of RcnAB in response to binding the non-cognate ion. The results are discussed within a protein allosteric model for gene regulation by metalloregulators.

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus (S. aureus) and is under the transcriptional control of the persulfide sensor CstR and H2S. Here we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (Ethylmalonic Encephalopathy Protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both activities require Cys408. CstB protects S. aureus against H2S toxicity with C201S and C408S cstB genes unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts the cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon the elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S stress, in concert with the other enzymes encoded by the cst operon.