DETERMINATION OF THE SELF-EXCHANGE RATE-CONSTANT FOR PLASTOCYANIN FROM ANABAENA-VARIABILIS BY NUCLEAR-MAGNETIC-RESONANCE LINE BROADENING

The rate constant (25-degrees-C) for self exchange between the copper-(I) and -(II) forms of Anabaena variabilis plastocyanin has been determined by an N M R method. The procedure used depends upon the enhanced relaxation of protons near the paramagnetic Cu(II) in the oxidised protein. At pH 7.5, I = 0.1 0 M the rate constant is 3.2 x 10(5) 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 x 10(5) M-1 s-1 under the same conditions. The rate constant is substantially (at least 10(2) 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.