Kinetic analysis of the metal binding mechanism of Escherichia coli manganese superoxide dismutase.
The acquisition of a catalytic metal cofactor is an essential step in the maturation of every metalloenzyme, including manganese superoxide dismutase (MnSOD). In this study, we have taken advantage of the quenching of intrinsic protein fluorescence by bound metal ions to continuously monitor the metallation reaction of Escherichia coli MnSOD in vitro, permitting a detailed kinetic characterization of the uptake mechanism. Apo-MnSOD metallation kinetics are "gated", zero order in metal ion for both the native Mn2+ and a nonnative metal ion (Co2+) used as a spectroscopic probe to provide greater sensitivity to metal binding. Cobalt-binding time courses measured over a range of temperatures (35-50 degrees C) reveal two exponential kinetic processes (fast and slow phases) associated with metal binding. The amplitude of the fast phase increases rapidly as the temperature is raised, reflecting the fraction of Apo-MnSOD in an "open" conformation, and its temperature dependence allows thermodynamic parameters to be estimated for the "closed" to "open" conformational transition. The sensitivity of the metallated protein to exogenously added chelator decreases progressively with time, consistent with annealing of an initially formed metalloprotein complex (k anneal = 0.4 min(-1)). A domain-separation mechanism is proposed for metal uptake by apo-MnSOD.
Whittaker
Mei M
MM
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006, USA.
Mizuno
Kazunori
K
Bächinger
Hans Peter
HP
Whittaker
James W
JW
D001703:Biophysics / Q000379:methods*; D002151:Calorimetry; D003035:Cobalt / Q000737:chemistry; D019281:Dimerization; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008670:Metals; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D008670:Metals; D003035:Cobalt; D008345:Manganese; D014364:Tryptophan; D004492:Edetic Acid; D013482:Superoxide Dismutase