Intramolecular electron transfer between [4Fe-4S] clusters studied by proton magnetic resonance spectroscopy

The rate constants for the intramolecular electron transfer between the two [4Fe-4S] clusters of a series of native and genetically engineered ferredoxins have been determined by proton magnetic resonance (1H NMR) spectroscopy. The measurement relies on the properties of the signals assigned to beta-protons of the coordinating cysteines when the protein is substoichiometrically reduced: these signals include coalesced peaks arising from the fast hopping of an extra electron between the two oxidized clusters of the protein. An upper limit of significantly less than 10(5) M(-1) s(-1) for the intermolecular and an average of the order of 5 x 10(6) s(-1) for the intramolecular electron transfer rate constants of several ferredoxins have been obtained. Owing to the edge-to-edge intercluster distance of approximately 10 A derived from the crystallographic structure of Clostridium acidurici ferredoxin, the rate constant associated with the intramolecular process is as expected for a nonadiabatic redox process, assuming a reasonable value of less than 1 eV for the reorganization energy. The latter could not 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 site-directed mutagenesis have been used to probe the potential involvement of dominant electron transfer pathways between the clusters. These changes have no major effect on the value of the intramolecular electron transfer rate constant. From this analysis, no specific amino acid side chain seems to play a central role in the process. The rate constants derived in the present work may serve as a basis for the study of enzymes containing two closely spaced [4Fe-4S] clusters such as found in these ferredoxins.