A 1H-NMR study on the blue copper protein amicyanin from Thiobacillus versutus. Resonance identifications, structural rearrangements and determination of the electron self-exchange rate constant.
A number of resonances in the 1H-NMR spectra of reduced and oxidised amicyanin from Thiobacillus versutus have been identified by one- and two-dimensional NMR techniques. The second-order electron self-exchange rate constant (8.5 x 10(4) M-1.s-1; pH = 7.4; T = 308.5 K) was determined by measuring the line broadening of six singlets in slightly oxidised solutions of the protein. A large increase in electron exchange rate is observed in the presence of ferrocyanide. The copper atom in the reactive centre of the protein appears to be coordinated by nitrogens from two histidines and sulfurs from a methionine and a cysteine. One of the ligand histidines becomes protonated at low pH [pK*a = 6.74 (+/- 0.02)], the asterisk indicating value uncorrected for the deuterium isotope effect] in reduced amicyanin. This is the first example of a non-photosynthetic blue copper protein in which a ligand histidine becomes protonated at low pH. A small pH-independent conformational rearrangement occurs upon oxidation.
Gorlaeus Laboratories, Leiden University, The Netherlands.
A
A
Lommen
G W
GW
Canters
J
J
van Beeumen
D000596:Amino Acids / Q000032:analysis; D001059:Apoproteins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D003300:Copper / Q000032:analysis; D004579:Electron Transport / Q000187:drug effects; D005292:Ferricyanides / Q000494:pharmacology; D005295:Ferrocyanides / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000502:physiology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013855:Thiobacillus / Q000032:analysis*
D000596:Amino Acids; D001059:Apoproteins; D001426:Bacterial Proteins; D005292:Ferricyanides; D005295:Ferrocyanides; D008667:Metalloproteins; C017079:mauC protein, Methylobacterium extorquens; C007931:hexacyanoferrate III; D003300:Copper; C020354:hexacyanoferrate II