Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichrois m spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalen t metal in its: active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increa ses the apparent stability of native RNase HI chiefly by reducing the unfol ding rate. As with the ape-form of the protein, refolding from high denatur ant concentrations in the presence of Mg2+ follows three-state kinetics: fo rmation of a rapid burst phase followed by measurable single exponential ki netics. Therefore, the overall folding pathway of RNase HI is minimally per turbed by the presence of metal ions. Our results indicate that the metal c ofactor enters the active site pocket only after the enzyme reaches its nat ive fold, and therefore, divalent metal binding stabilizes the protein by d ecreasing its unfolding rate. Furthermore, the binding of the cofactor is d ependent upon a carboxylate critical fur activity (Asp10). A mutation in th is residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in th e presence of metal.