The NDH was activated by illuminating the thylakoids in the presence of the assay reagents for 45 min before measurement. Even though the light-dependent assay eliminates the interference from mitochondrial complex I, we resolved to quantitate Ki8751 the degree of mitochondrial contamination. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible. The genomes of cyanobacteria and most plant chloroplasts contain 11 genes (gene products form a complex that can be isolated from thylakoid membranes (Funk and Steinmller, 1995; Sazanov et al., 1995; Ki8751 Quiles et al., 1996). In bacteria (other than cyanobacteria) complex I consists of 14 subunits that are also conserved in mitochondrial complex I (Friedrich et al., 1995). The cyanobacterial and chloroplast NDH seem to lack homologs of the three essential subunits that constitute the NADH-oxidation site in the bacterial and Rabbit Polyclonal to CEP78 mitochondrial complex (Grohman et al., 1996). The function of NDH in chloroplasts is not understood, but a role in cyclic electron transport and/or chlororespiration would seem to be likely. A role in cyclic electron transport would imply electron donation from stromal NADPH via Ki8751 the membrane-bound NDH complex to the plastoquinone pool. Kubicki et al. (1996) showed that in sorghum the genes are preferentially expressed in bundle-sheath chloroplasts, the apparent site of cyclic electron flow in C4 species. Thus, this finding is in agreement with a function of the NDH complex in cyclic electron transport. NDH activity has been demonstrated in the thylakoid membranes of several different species of plants, algae, and cyanobacteria (Mi et al., 1992a, 1992b, 1994, 1995; Yu et al., 1993; Cuello et al., 1995; Sazanov et al., 1995; Quiles et al., 1996; Seidel-Guyenot et al., 1996). However, direct demonstration of an involvement in cyclic electron transport in most cases has not been achieved. Mi et al. (1995), working with mutants of mutants of sp. PCC 7002 were not deficient in cyclic electron transport (Yu et al., 1993). Cyclic electron transport via NDH is most easily understood if the complex can use NADPH, as shown by Mi et al. (1995). However, there is a lack of consensus on the substrate specificity of the NDH complex. In barley (and sp. PCC 6803 (Mi et al., 1995), different specificities have been reported for other species (for review, see Schmetterer, 1994). It is futile to look for a unifying principle covering all oxygenic phototrophs. It seems likely that NDH is involved in both cyclic and respiratory pathways, and that its relative contribution to different pathways may differ between species or even within a species dependent on growth conditions. The issue of specificity is complicated by the different assay conditions used, by mitochondrial contamination, and by interference from NADPH oxidation by FNR. In most previous studies the dehydrogenase activities have been assayed in the dark with artificial acceptors such as ferricyanide or soluble quinones. Both mitochondrial complex I and FNR will show high activity in such assays. In this paper we have used a light-specific assay that eliminates the interference from contaminating activities, and we clearly demonstrate an NAD(P)H dehydrogenation that functions with equal efficiency with both substrates. A further unsolved question is which subunit(s) contains the NAD(P)H-binding site of the NDH complex? The chloroplast genome has no homolog of the NADH-binding flavoprotein of complex I, and evidence against the presence of a nuclear-encoded chloroplast homolog has been presented (Grohman et al., 1996). The genome of sp. PCC 6803 contains open reading frames in the hydrogenase operon with some similarity to the NADH-oxidizing subunits in other bacteria (Appel and Schulz, 1996). Whether the gene products are part of cyanobacterial NDH remains to be shown. Quiles et al. (1996) reported the presence Ki8751 of a 53-kD NADH-oxidizing protein in barley chloroplasts, and have suggested that this protein could be a component of the NDH complex. The 53-kD protein was specific for NADH rather than NADPH. Guedeney et al. (1996) showed that the flavoprotein FNR binds to several polypeptides of the NDH in tobacco thylakoids, and have suggested that FNR in thylakoids could be the functional equivalent of the NADH-oxidizing domain in complex I. This could explain the result of Mi et al..