Mutations of Glu92 in Ferredoxin I from Spinach Leaves Produce Proteins Fully Functional in Electron Transfer but Less Efficient in Supporting NADP⁺ Photoreduction

Abstract

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP' reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH-cytochrome c reductase reaction catalysed by ferredoxin-NADP' reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP' photoreduction than the wild-type protein: both V and the apparent K, for reduced ferredoxin were influenced. On the other hand, the K', values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-+Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I [Aliverti, A,, Hagen, W. R. & Zanetti, G. (1995) FEBS Lett. 368, 220-2241, Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.

Residue Glu‐91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin‐thioredoxin reductase

“…Our results strongly suggest that in the C terminus, only residue Glu-9l (E92 in spinach) is essential for this interaction. The results obtained in the present study also fit very well with the data of [8] where mutations of the E92 residue of spinach ferredoxin was found to increase its reactivity with FNR in the cytochrome c reduction assay. On the other hand, the triple mutant in the al helix is completely inactive in the NADP-MDH light activation assay, a feature which was not expected from the results in [10].…”

“…On the other hand, the Glu-91 mutants of Chlamydomonas reinhardtii are indeed reduced by PSI since the (E91K) mutant supports NADP photoreduction, albeit at a reduced rate (ca. 50%, data not shown), in a similar manner to the corresponding (E92K) mutant of spinach [8]. It is highly unlikely that the incapacity of the Glu-91 mutants to promote the NADP-MDH activation results from the poorer interaction with PSI since the process of enzyme light activation requires a much lower electron flow than NADP photoreduction [21].…”

“…So far, the most studied interaction of ferredoxin is with its associated enzyme ferredoxin NADP oxidoreductase (FNR), a protein involved in the NADP reduction in the chloroplast stroma. Contrasting results have been obtained by site directed mutagenesis, Hurley et al [7] reporting a 20000-fold decrease in the second order rate constant for electron transfer to FNR for the mutant (E94K) in Anabaena, and Piubelli et al [8] finding only moderate effects of the equivalent mutation (E92K) in spinach. However, the two papers did not measure the same reaction, one utilizing flash laser spectroscopy, and the other cytochrome c reduction, which makes the results more difficult to compare.…”

Stearoyl-Acyl Carrier Protein and Unusual Acyl-Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin

“…strain PCC 7119 and spinach and found that electrostatic interactions most likely contributed to the formation of protein complex that permits electron transfer. Furthermore, it has been determined that alterations at specific positions (E92) in spinach ferredoxin could increase activity of ferredoxin in supporting NADPH/FNR mediated cyt C reduction and reduced the activity of ferredoxin in supporting photoreduction of NADP ϩ by photosystem I (Piubelli et al, 1996). These studies collectively show it is possible to alter kinetic activity without significantly altering redox potential.…”

“…Redox potential of Anabaena sp. ferredoxin has been determined to be Ϫ440 mV (Hurley et al, 1993), whereas the major leaf isoform (Fd I) in spinach has a redox potential measured at Ϫ400 mV (Aliverti et al, 1995;Piubelli et al, 1996). Since NADPH/NADP ϩ redox potential is Ϫ320 mV the transfer of electrons from NADPH to the Anabaena sp.…”