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

Abstract.

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 Mössbauer signals, but ΔE _Q (1.1 mm/s) and δ (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mössbauer experiments, occurs with a measured rate constant k _obs≈0.72±0.35 s^–1 that is consistent with results from time-resolved electronic absorption and fluorescence (k _obs≈0.4±0.1 s^–1) and EPR (k _obs≈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≈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, Mössbauer and EPR measurements.