A chimeric iron-sulfur flavoprotein endowed with NADPH-cytochrome c reductase activity.

Abstract

Ferredoxin-NADP' reductase (FNR) catalyses the last step in the photosynthetic electron transport chain: it pairs single electrons carried by ferredoxin (Fd) and transfers them as a hydride to NADP'. FNR is the prototype of a large family of flavin-dependent enzymes that function in various electron transport chains as transducers between oneand two-electron carriers [ 1, 21. Besides the flavin prosthetic group, these enzymes have additional reducible cofactors, which can reside in linked domains or in dissociable protein units, as is the case for the FNRiFd couple. FNR and Fd contain FAD and a [2Fe-2S] cluster, respectively. Both proteins from spinach leaves have been cloned and expressed in E. coli [3, 41. The three-dimensional structures of spinach FNR [5] and of cyanobacterial Fd are known at high resolution. To provide a new tool for studying the interaction between Fd and FNR, a chimeric protein, comprising the two functional units on the same polypeptide chain, was designed. The chimeric protein should acquire a domain organisation resembling that of known members of the family, such as phthalate dioxygenase reductase and oxygenase reductase, where a Fd-like domain is linked to the basic FNR twodomain motif [4]. A gene construct encoding the chimeric three-domain protein has been obtained by fusion of the 5' end of the cDNA part coding for the mature spinach FNR to the 3' end of the cDNA fragment encoding the mature isoform I of spinach leaf Fd (Fd I). This arrangement was chosen to take advantage of the fact that the first 18 residues of FNR do not have an ordered structure, thus providing a natural spacer arm between the folding units. This construct has been inserted in the 'vector PET-lld and overexpressed in E. coli. The recombinant protein was synthesised in the bacterial host at a level of about 1% of the soluble proteins. The chimera has been purified with an overall yield of about 45% by a combination of the procedures used to isolate the individual proteins. In non-denaturing PAGE, the fusion protein showed an electrophoretic mobility intermediate between those of FNR and Fd I. The apparent M, of the chimeric protein, as measured by 0.4 L I i i 1 i I I I I i I I I I I I i i I 1 1