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Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to siroheme reduction with the leaf enzyme, whereas the siroheme and the [4Fe-4S} cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s-1) and for kcat (420-450 electrons per second and per nitrite reductase). When nitrite reductase is initially pre-reduced to the state ferrous siroheme-NO., the fast kinetics of reduction by ferredoxin and the thermodynamics of ferredoxin binding are equivalent to those observed with oxidized nitrite reductase without nitrite. Spectral and kinetic analyses of single reduction of the recombinant enzyme in the ferrous siroheme-NO. state by photoreduced ferredoxin reveal that this process leads to reduction of the [4Fe-4S] cluster with little, if any, NO. reduction. These data show that the enzyme must wait for the next reduction step before NO. undergoes substantial reduction. [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 flash photolysis study of electron transfer (ET) kinetics from reduced ferredoxin (photoreduced by Photosystem I) to the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been carried out. In the presence of nitrate, under conditions where only a single electron is transferred to nitrate reductase, the rate of enzyme reduction shows a biphasic concentration dependence: At low enzyme concentrations the dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 x 107 M-1 s-1; at concentrations above 2 µM, the rate increases nonlinearly to an asymptotic value of approximately 300 s-1, indicating the presence of a rate-limiting step in the process. The spectrum of the one-electron reduced enzyme suggests that Mo centers are largely reduced with a minor contribution of iron--sulfur cluster reduction. Under conditions favoring two-electron reduction of the enzyme, the redox difference spectrum can be accounted for by the oxidation of two reduced ferredoxins, suggesting that the enzyme has completed one full catalytic cycle. The spectral changes observed in the absence of nitrate are significantly different from those seen in the presence of nitrate. Experiments in the absence of nitrate revealed that the singly reduced enzyme exhibits different absorption characteristics and reoxidation kinetics, compared to those observed with nitrate present, and exhibits a much faster binding by reduced ferredoxin than the oxidized enzyme. The implications of these observations for understanding the enzyme mechanism are discussed. [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]