Intramolecular Electron Transfer between [4Fe-4S] Clusters Studied by Proton Magnetic Resonance Spectroscopy

Abstract

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.

Intramolecular electron transfer in [4Fe-4S] proteins: estimates of the reorganization energy and electronic coupling in Chromatium vinosum ferredoxin

“…2), arising from the dynamic equilibrium between two [4Fe-4S] clusters rapidly exchanging one electron, are broadened compared to previously characterized samples of the protein, including the native form. The intramolecular ET rate constant, k, can be derived from this NMR line-broadening and is the sum of k f , the ET rate constant from cluster I to cluster II, and k ±f , associated with the reverse process [20,21]. The ratio of these individual rates depends on the ratio of the oxidized and reduced molar fractions of the exchanging clusters [20,21,23]; these parameters can be calculated either from the electrochemical data (Table 1) [20].…”

“…The procedure to extract ET rates from line broadening measurements in the case of 2[4Fe-4S] ferredoxins has been detailed elsewhere [20]. Briefly, the linewidth G sr of the coalesced (i.e.…”

“…However, these additional data cannot be used to improve the precision of the measurements, as they are less accurate than those obtained with Cys18 Hb 2 . Kinetically significant intermolecular ET among ferredoxin molecules at different redox levels is needed to observe EXSY signals, but the corresponding rate constants are far smaller than those of intramolecular ET, as discussed elsewhere [17,20].…”

“…Each [4Fe-4S] cluster can exchange one electron and, when substoichiometric reducing equivalents are added to the oxidized ferredoxin, electron hopping occurs between the two clusters, as observed with NMR spectroscopy [17,18,19,20,21]. Rates of intramolecular ET can be derived from analysis of coalesced spectral lines [20,21].…”

“…Rates of intramolecular ET can be derived from analysis of coalesced spectral lines [20,21]. This analysis has up to now been limited to isopotential ferredoxins [20,21], since the rates in C. vinosum ferredoxin, with a large driving force between the clusters, exceeded the kinetic window of NMR [22]. This limitation has now been lifted by use of new derivatives, which has allowed us to fit the experimental rates to the semiclassical form of the standard ET theory.…”