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 k(cat) (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.

The electron transfer cascade from photosystem I to NADP ؉ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP ؉ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP ؉ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s ؊1 ) and the redox potential of the first reduction of ferredoxin-NADP ؉ reductase (؊380 mV) were determined. In the absence of NADP ؉ , differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP ؉ reductase. An effective rate of 140 -170 s ؊1 was also measured for the second reduction of ferredoxin-NADP ؉ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP ؉ , the second-order rate constant for the first reduction of ferredoxin-NADP ؉ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP ؉ reductase)-NADP ؉ -ferredoxin. A single catalytic turnover was monitored, with 50% NADP ؉ being reduced in 8 -10 ms using 1.6 M photosystem I. In conditions of multiple turnover, we determined initial rates of 360 -410 electrons per s and per ferredoxin-NADP ؉ reductase for the reoxidation of 3.5 M photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP ؉ photoreduction.