Circular dichroism, binding and immunological studies on the interaction between spinach ferredoxin and glutamate synthase

Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism

“…PCC 6803 ferredoxin/Fd-glutamate synthase complex, other cross-linking experiments support a stoichiometry of two ferredoxins:one Fd-glutamate synthase for the complex formed by the spinach proteins ) and the proteins from the green algae C. reinhardtii (Garcı´a-Sa´nchez et al 2000) and Monoraphidium braunii (Vigara et al 1996). Membrane ultrafiltration experiments with the spinach proteins are also more consistent with a 2:1 ferredoxin:Fd-glutamate synthase stoichiometry for the complex (Hirasawa et al 1989). As the stoichiometry of the complex has important implications for the enzyme mechanism (i.e., in deciding whether the two electrons required for the reaction arrive separately in two sequential electron transfers from reduced ferredoxin or whether it may be possible that a concerted transfer of two electrons from two simultaneously bound ferredoxins might occur), it is of considerable importance to remove uncertainties as to the correct value.…”

“…PCC 7942 (Hirasawa et al 2004) the ferredoxin/enzyme stoichiometry of the complexes has been shown to be 1:1. Immunological experiments support the idea that the ferredoxin-binding site on spinach Fd-glutamate synthase resembles the ferredoxin-binding site on FNR and on nitrite reductase (Hirasawa et al 1989 and thus it seemed reasonable to expect that the stoichiometry of the ferredoxin/ Fd-glutamate synthase complex would also be 1:1. However, while there is evidence from cross-linking (Schmitz et al 1996), small angle X-ray scattering (van den ) and mass spectrometry (van den Heuvel et al 2004) experiments supporting a 1:1 stoichiometry for the Synechocystis sp.…”

“…These spectral perturbations have been interpreted as arising from conformational changes in one or both proteins that result from complex formation (Hirasawa et al 1989). Changes in CD spectra have also been observed as a consequence of complex formation between ferredoxin and both FNR and nitrite reductase .…”

The Ferredoxin-Binding Site of Ferredoxin:Nitrite Oxidoreductase (Differential Chemical Modification of the Free Enzyme and Its Complex with Ferredoxin)

“…The data presented, implicating R375, R556, and K436 as likely to be present at or near the Fd-binding site of nitrite reductase, to our knowledge represent the first direct evidente as to the location of this substrate-binding site on a key enzyme of plant nitrogen metabolism. Although immunological evidence suggests that the positively charged, Fd-binding domain on FNR is likely to share some common features with the positively charged, Fd-binding domain on nitrite reductase (Hirasawa et al, 1989), no significant sequence homology could be detected between any of the Arg-containing or Lys-containing nitrite reductase peptides described above and regions of FNR that have been implicated (Knaff, 1996) in Fd-binding. It may be that there are other regions in the nitrite reductase primary structure that form part of the three-dimensional structure of the Fd-binding site of this enzyme, which shows sequence homologies to regions of FNR, and that these regions simply have not been detected by the differential chemical modification studies described above.…”

“…Because evidence exists suggesting that the binding of Fd to spinach nitrite reductase is similar to that involved in the complex formation between Fd and FNR (Hirasawa et al, 1986(Hirasawa et al, , 1989, it seemed appropriate to apply this technique to mapping the binding site for Fd on nitrite reductase. Here we present the results of such a study, which has provided evidence for the sequence positions of two Arg residues and one Lys residue at the Fd-binding site of nitrite reductase.…”