The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry.
The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa(red)=6.3 and pKa(ox) < or = 3.2 at 22 degrees C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s-1 at 25 degrees C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 degrees C, at which the rate constant for this conversion was determined to be 35 +/- 20 s-1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.
Jeuken
Lars C
LC
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
Camba
Raul
R
Armstrong
Fraser A
FA
Canters
Gerard W
GW
D001426:Bacterial Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008667:Metalloproteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000378:metabolism*; D011199:Potentiometry / Q000379:methods*; D013816:Thermodynamics
D001426:Bacterial Proteins; D008667:Metalloproteins; C017079:mauC protein, Methylobacterium extorquens; D006639:Histidine; D003300:Copper