Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP(+) photoreduction

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-m for reduced ferredoxin were influenced. On the other hand, the K-d 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, 220-224]. 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.