Determination of the self-exchange rate constant for plastocyanin from Anabaena variabilis by nuclear magnetic resonance line broadening
Abstract
The rate constant (25 °C) for self exchange between the copper-(I) and -(II) forms of Anabaena variabilis plastocyanin has been determined by an NMR method. The procedure used depends upon the enhanced relaxation of protons near the paramagnetic CuII in the oxidised protein. At pH 7.5, /= 0.10 M the rate constant is 3.2 × 105 M–1 S–1 and provides confirmation of that obtained indirectly by application of the Marcus equation to the cross-reaction of azurin [ACu(I)] with A. variabilis plastocyanin [PCu(II)]. This gives 5.9 × 105 M–1 s–1 under the same conditions. The rate constant is substantially (at least 102 times) larger than those obtained for higher-plant plastocyanins, e.g. spinach, and this is attributable to the higher negative charge on the latter. Since the rate constant is comparable to that for azurin itself a similar mechanism for self exchange involving association of protein molecules via hydrophobic adjacent surfaces is suggested.