Laser flash photolysis studies of the kinetics of reduction of spinach and Clostridium ferredoxins by a viologen analog: electrostatically controlled nonproductive complex formation and differential reactivity among the iron-sulfur clusters

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Abstract
We have studied the transient kinetics of electron transfer from a positively charged viologen analogue (propylene diquat), reduced by pulsed laser excitation of the deazariboflavin/EDTA system, to the net negatively charged ferredoxins from spinach and Clostridium pasteurianum. Spinach ferredoxin showed monophasic kinetics over the ionic strength range studied, consistent with the presence of only a single iron-sulfur center. Clostridium ferredoxin at low ionic strength showed biphasic kinetics, which indicates a differential reactivity of the two iron-sulfur centers of this molecule toward the electron donor. The kobsd values for the initial fast phase observed with Clostridium ferredoxin were ionic strength dependent, whereas the slow-phase kinetics were ionic strength independent. This correlates with the highly asymmetric charge distribution on the surface of the bacterial protein relative to the two iron-sulfur clusters. The kinetics corresponding to spinach ferredoxin reduction were also ionic strength dependent, and the results obtained with these kinetics and with the fast phase of the bacterial ferredoxin reduction were consistent with a mechanism involving electrostatistically stabilized complex formation. For spinach ferredoxin, the second-order rate constant extrapolated to infinite ionic strength was 2-fold smaller, and extrapolated limiting first-order rate was 10-fold smaller, than for Clostridium ferredoxin, indicating a smaller intrinsic reactivity of the spinach protein toward the electron donor. Differences in the rate constant values and the ionic strength dependencies with both ferredoxins are consistent with differences in cluster structure and environment and protein size and charge distribution. For both proteins, the total amount of ferredoxin reduced increased with the ionic strength. This can be explained by assuming more than one electrostatic interaction site on the protein, leading to both productive and nonproductive interactions with the electron donor. This explantation is also consistent with effects observed upon the addition of a positively charged polypeptide (polylysine) to the reaction mixture, which decreased the reaction rate constant by competing with the electron donor for the productive site, while increasing the total amount of reduced ferrodoxin by also competing for the nonproductive sites. Polylysine had a much smaller effect on Clostridium ferredoxin reduction as compared with the spinach protein. This is again consistent with the differences in size and charge distributions of the two proteins.

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