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

To investigate the structural control mechanisms in the metal site of amicyanin when interacting with MADH, redox-inactive Ag+ - and Cd2- substituted amicyanins were studied with perturbed angular correlations of alpha-rays (PAC) spectroscopy. PAC experiments on 111mCd-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(plus-minus)2 (mu)M with MADHred and 38(plus-minus)11 (mu)M with MADHox, indicating that the Cd-amicyanin binding affinity is regulated by the MADH redox state. PAC experiments on 111Ag-substituted amicyanin also showed two different forms of g-Aamicyanin, probably reflecting relaxation from Ag to Cd geometry. No binding of g-Aamicyanin to MADH could be observed with PAC, suggesting that the K d is larger than 43 (mu)M, 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 away from the metal ion. The Ag+- and Cd2- 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 pK 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.