Elucidation of a [4Fe-4S] cluster degradation pathway:: rapid kinetic studies of the degradation of Chromatium vinosum HiPIP

Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-potential iron protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened Mossbauer signals, but DeltaE(O) (1.1 mm/s) and delta (0.34 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mossbauer experiments, occurs with a measured rate constant k(obs) approximate to 0.72+/-0.35 s(-1) that is consistent with results from time-resolved electronic absorption and fluorescence (k(obs) approximate to 0.4+/-0.1 s(-1)) and EPR (k(obs) approximate to 0.62+/-0.18 s(-1)) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the protein, facilitating protonation of the cluster, but not degradation per se. Release of iron ions occurs even more slowly with k(obs) approximate to 0.07+/-0.02 s(-1) as determined by the appearance of the g=4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, Mossbauer and EPR measurements.