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Kinetic Analysis of the Metal Binding Mechanism of Escherichia coli Manganese Superoxide Dismutase

Mei M. Whittaker ^*, Kazunori Mizuno  , Hans Peter Bächinger   and James W. Whittaker ^*

^* Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006; Department of Research, Shriners Hospital for Children, Portland, Oregon 97239; and Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97201

Correspondence: Address reprint requests to James W. Whittaker, Dept. of Environmental and Biomolecular Systems, Oregon Health and Science University, 20000 NW Walker Rd., Beaverton, Oregon 97006. Tel.: 503-748-1065; Fax: 503-748-1464; E-mail: jim{at}ebs.ogi.edu.

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°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.