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 Cu^II 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.