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This paper reports on extensive molecular dynamics simulations (about 40 ns in total) in both the reduced and the oxidized states of Ferredoxin from Cyanobacterium Anabaena PCC71 19. These calculations have provided us with the free energy profile of the φ[SUB47] backbone angle which controls the "CO in" to "CO out" transition of Cys46 in the reduced and oxidized Fd7119. Our main motivation has been to identify the time scales involved in the reduction of Fd and single out the amino acid residues crucially affecting the conformational change and, thus, electron transfer. The free energy profiles obtained in this study are relevant to electron transfers in the PSI/Fd71 19 and Fd71 1 9/FNR complexes. Our findings based on hydrated ferredoxin simulations are that activated processes are to occur in the protein during electron transfer to and from ferredoxin. The relative stability and the activation barrier of the "CO in" to "CO out" transition can be modulated by the distance between the Ser47 and the G1u94 residues. In our calculations, for short distances, the "CO in" state is favored in the reduced form, whereas for large distances, the "CO out" state becomes increasingly favored. Accordingly, conformational changes in Fd71 19 when bound to PSI or FNR can have crucial effects on the kinetics of the electron transfer. Our simulations also show that the hydrogen bond between between Ser47(OG) and Cys46(O) is essential to lock in the "CO out" state. This finding explains why only the Ser47Thr Fd7119 mutant sustains electron transfer activity, as only residues serine and threonine can form a specific hydrogen bond with Cys46(O). Finally, our simulations predict that Phe65 has a large probability of being in close contact with the Cys46(O) at the top of the conformational free energy barrier. [ABSTRACT FROM AUTHOR]

The dissociation constant κd of the photosystem I (PSI): ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. κd values of 0.14-0.38 μM were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection. [ABSTRACT FROM AUTHOR]

A multi-enzyme electron-transfer chain involving solubilized photosystem I (PSI) as photocatalytic unit, cytochrome c6 and ferredoxin as electron carriers and ferredoxin/NADPH oxidoreductase (FNR) as electron acceptor was reconstituted in an electrochemical cell and studied by cyclic voltammetry. The working gold electrodes were modified to react selectively with cytochrome C6. Quantitative analysis of the photocatalytic current under continuous illumination allowed the determination of the values kon and koff for the ferredoxin/PSI interaction. An efficient recycling system for NADPH was established, and the dissociation constant of the oxidized ferredoxin/semiquinone FNR complex was extracted by modeling the catalytic efficiency of the chain as a function of ferredoxin concentration. The value determined hereby is consistent with a shift of -50 to -100 mV of the reduction potential of ferredoxin when complexed with FNR. [ABSTRACT FROM AUTHOR]

A light-activated electron-transfer chain was assembled using solubilized cyanobacterialphotosystem I as photoactive enzyme, cytochrome 06 (also from cyanobacteria) as electron donor, andmethyl viologen as electron acceptor. The photocatalytic activity of the ensemble was measured by directand reversible electrochemistry of cytochrome 06 at a surface-modified gold electrode. Analysis of theelectrochemical response with an appropriate model for the reaction mechanism allowed the relation of the overall catalytic reaction rate to the individual steps of the catalytic cycle. Second-order rate constants were determined for the first time under steady-state conditions. The results validate this approach as an efficient method for the study of electron transfer between photoactive enzymes and their redox partners. [ABSTRACT FROM AUTHOR]