Amicyanin metal-site structure and interaction with MADH: PAC and NMR spectroscopy of Ag-, Cd-, and Cu-amicyanin.

To investigate the structural control mechanisms in the metal site of amicyanin when interacting with MADH, redox-inactive Ag(+)- and Cd(2+)-substituted amicyanins were studied with perturbed angular correlations of gamma-rays (PAC) spectroscopy. PAC experiments on (111m)Cd-substituted amicyanin revealed two different metal-site structures, which are very likely in dynamic exchange on a ~5 ns timescale. Only one structure binds to MADH. The dissociation constants, K(d), are 9+/-2 microM with MADH(red) and 38+/-11 microM with MADH(ox), indicating that the Cd-amicyanin binding affinity is regulated by the MADH redox state. PAC experiments on (111)Ag-substituted amicyanin also showed two different forms of Ag-amicyanin, probably reflecting relaxation from Ag to Cd geometry. No binding of Ag-amicyanin to MADH could be observed with PAC, suggesting that the K(d) is larger than 43 microM, based on the 95% confidence limit. NMR revealed large chemical shift differences between native copper amicyanin and both metal-substituted forms. Affected residues are found up to 15 A away from the metal ion. The Ag(+)- and Cd(2+)-substituted amicyanins demonstrate no change in coordination as a function of pH, contrary to Cu(+)-amicyanin which shows protonation of the copper ligand His96 with p K(a)=6.8. It is concluded that, contrary to other blue copper proteins, Ag(+)-amicyanin is not a close mimic of Cu(+)-amicyanin, and that structural changes in the metal site have large effects on the affinity for the redox partner.