Direct monitoring of metal ion transfer between two trafficking proteins.
To avoid the toxic effects of both essential and nonessential metal ions, cells elaborate a variety of metal ion trafficking proteins that regulate metal ion mobility. While structures of several trafficking proteins have been determined, kinetic characterization of metal ion transfer between cognate protein partners and the factors controlling transfer has lagged behind, in part due to a limitation on methods to monitor the rapid transfer. In this Communication we report studies on the kinetics of Hg2+ transfer between separately expressed components of the flavoenzyme mercuric ion reductase (MerA) that take advantage of the sensitivity of the flavin fluorescence to the charge state of a cysteine thiol in the Hg2+ binding pathway. The thiolate form of C558 in the Tn501 MerA partially quenches the fluorescence of the oxidized enzyme. Protonation or binding of Hg2+ to the thiolate increases the fluorescence, providing a sensitive probe for kinetic analysis of the Hg2+ binding reaction. The kinetics of Hg2+ transfer in both directions between the cysteine pair of the separately expressed N-terminal domain and the C-terminal cysteines (C558, C559) of the catalytic core are presented, along with a model describing the overall process.
Ledwidge
Richard
R
Department of Pharmaceutical Chemistry, 600 16th Street, University of California-San Francisco, San Francisco, CA 94143-2280, USA.
Soinski
Rebecca
R
Miller
Susan M
SM
D007700:Kinetics; D008670:Metals / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
D008670:Metals; D011506:Proteins; D010088:Oxidoreductases; C021551:mercuric reductase