title: Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA.
abstract: In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
authors.forename: Gopal R
authors.initials: GR
authors.lastname: Periyannan
authors.forename: Alison L
authors.initials: AL
authors.lastname: Costello
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Ke-Wu
authors.initials: KW
authors.lastname: Yang
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D003035:Cobalt; C088032:beta-lactamase L1; C111082:ImiS protein, Aeromonas veronii; D001618:beta-Lactamases; C063912:carbapenemase