Copper binding before polypeptide folding speeds up formation of active (holo) Pseudomonas aeruginosa azurin

Cofactors often stabilize the native state of the proteins; however, their effects on folding dynamics remain poorly understood. To uncover the role o f one cofactor, we have examined the folding kinetics of Pseudomonas aerugi nosa azurin, a small blue-copper protein with a copper cofactor uniquely co ordinated to five protein residues. Copper removal produces apo-azurin whic h adopts a folded structure identical to that of the holo-form. The folding and unfolding kinetics for apo-azurin follow two-state behavior. The extra polated folding time in water, tau similar to 7 ms, is in good agreement wi th the topology-based prediction. Copper uptake by folded apo-azurin, to go vern active (holo) protein, is slow (tau similar to 14 min, 50:1 copper-to- protein ratio). In contrast, the formation of active (holo) azurin is much faster when copper is allowed to interact with the unfolded polypeptide. Re folding in the presence of 10:1, 50:1, and 100:1 copper:protein ratios yiel ds identical time-trajectories: active azurin forms in two kinetic phases w ith folding times, extrapolated to water, of tau = 10 +/-2 ms (major phase) and tau = 190 +/- 30 ms (minor phase), respectively. Correlating copper-bi nding studies, with a small peptide derived from the metal-binding region o f azurin, support that initial cofactor binding is fast (tau similar to 3.7 ms) and thus not rate-limiting. Taken together; introducing copper prior t o protein folding does not speed up the polypeptide-folding rate; neverthel ess, it results in much faster (> 4000-fold) formation of active (i.e., hol o) azurin. Living systems depend on efficient formation of functional biomo lecules; attachment of cofactors prior to polypeptide folding appears to be one method to achieve this.