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.
Jørgensen
Lars Elkjaer
LE
Department of Mathematics and Physics, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
Ubbink
Marcellus
M
Danielsen
Eva
E
D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism; D010230:Paracoccus / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D012834:Silver / Q000737:chemistry*; D013051:Spectrometry, Gamma
D002352:Carrier Proteins; D008667:Metalloproteins; C023548:copper-binding protein; D002104:Cadmium; D012834:Silver; D003300:Copper; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors