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Analysis of SlyD, A New Component of Hydrogenase Metallocenter Assembly in E. coli Deborah B. Zamble, Department of Chemistry, University of Toronto As with many metalloenzymes, the biosynthesis of the [NiFe] hydrogenase is a multi-step process in which accessory proteins assemble the intricate active site of the enzyme. An understanding of this complicated molecular pathway is essential in order to realize the potential of hydrogenase enzymes for biotechnological applications or antibiotic targets, and will contribute to our knowledge about intracellular metal homeostasis, a fundamental aspect of life. The E. coli protein SlyD is a peptidyl-prolyl isomerase with a C-terminal domain rich in metal-binding amino acids. SlyD forms a complex with the hydrogenase accessory protein HypB and contributes to nickel insertion into the hydrogenase precursor protein. By using a combination of in vitro and in vivo methods we are studying the biological chemistry of this new component of nickel metabolism. These experiments demonstrate that: • In addition to being a peptidyl-prolyl isomerase, SlyD acts as a protein folding chaperone. • The folding chaperone motif in SlyD and the metal-binding domain are both essential in vivo, whereas the peptidyl-prolyl isomerase activity is not required. • Mutating the folding chaperone motif, but not the metal-binding domain, impairs the interaction with HypB. • SlyD activates metal release from a Ni(II)-HypB complex. This acitivty requires the metal-binding domain of SlyD but not the peptidyl-prolyl isomerase activity. • SlyD binds up to 6 nickel ions with ranges of affinity that cover more than 7 orders of magnitude. HypE ? HypC HypD HypF SlyD Ni2+ N C HypC Fe2+ C O HycE HypA HypB C N CO2- GTP ? HypB Ni2+ N HypA C Fe2+ C O HypC C HycE GDP+Pi N N Ni2+ C 2+ Fe C C HycE O N CO2- CO2HycI CO + HH2O2 + + 2H CO 2 + +2 H2e+ 2 e Active Hydrogenase