INTRAMOLECULAR ELECTRON-TRANSFER BETWEEN [4FE-4S] CLUSTERS STUDIED BYPROTON MAGNETIC-RESONANCE SPECTROSCOPY

The rate constants for the intramolecular electron transfer between th e two [4Fe-4S] clusters of a series of native and genetically engineer ed ferredoxins have been determined by proton magnetic resonance (H-1 NMR) spectroscopy. The measurement relies on the properties of the sig nals assigned to beta-protons of the coordinating cysteines when the p rotein is substoichiometrically reduced: these signals include coalesc ed peaks arising from the fast hopping of an extra electron between th e two oxidized clusters of the protein. An upper limit of significantl y less than 10(5) M-1 s(-1) for the intermolecular and an average of t he order of 5 x 10(6) s(-1) for the intramolecular electron transfer r ate constants of several ferredoxins have been obtained. Owing to the edge-to-edge intercluster distance of approximately 10 Angstrom derive d from the crystallographic structure of Clostridium acidurici ferredo xin, the rate constant associated with the intramolecular process is a s expected for a nonadiabatic redox process, assuming a reasonable val ue of less than 1 eV for the reorganization energy. The latter could n ot be determined from the temperature dependence of the rate constant since no variation was observed over the temperature range accessible in these experiments, Structural changes introduced around and between the two [4Fe-4S] clusters in Clostridium pasteurianum ferredoxin by s ite-directed mutagenesis have been used to probe the potential involve ment of dominant electron transfer pathways between the clusters, Thes e changes have no major effect on the value of the intramolecular elec tron transfer rate constant. From this analysis, no specific amino aci d side chain seems to play a central role in the process, The rate con stants derived in the present work may serve as a basis for the study of enzymes containing two closely spaced [4Fe-4S] clusters such as fou nd in these ferredoxins.