Mw. Foster et al., Elucidation of a [4Fe-4S] cluster degradation pathway: rapid kinetic studies of the degradation of Chromatium vinosum HiPIP, J BIOL I CH, 6(3), 2001, pp. 266-274
Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-p
otential iron protein (HiPIP) has been investigated in the presence of a lo
w concentration of guanidinium hydrochloride. From the dependence of degrad
ation rate on [H+], it is deduced that at least three protons are required
to trigger efficient cluster degradation. Under these conditions the proton
ated cluster shows broadened Mossbauer signals, but DeltaE(O) (1.1 mm/s) an
d delta (0.34 mm/s) are similar to the native form. Collapse of the protona
ted transition state complex, revealed by rapid-quench Mossbauer experiment
s, 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 absorpti
on 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 hy
drochloride serves to perturb the tertiary structure of the protein, facili
tating protonation of the cluster, but not degradation per se. Release of i
ron 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, wi
th 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 meas
urements.