Enzymic Mechanisms of Pyridine Nucleotide Reduction in Chloroplasts

Abstract

The photochemical reduction of triphosphopyridine nucleotide by isolated chloroplasts has been known since 1951 (see “Discussion”). The discovery of this photochemical reaction marked a significant advance in the biochemical analysis of the energy conversion process of photosynthesis because it established the first link between photon capture by chloroplast pigments and the reduction of a physiological electron carrier with a strongly electronegative oxidation-reduction potential (E’o = -0.320 volt, pH 7). Before 1951, isolated chloroplasts were known to reduce only nonphysiological electron acceptors (Hill reagents) with electropositive oxidation-reduction potentials, e.g. ferricyanide and benzoquinone. It was generalized, therefore, that illuminated chloroplasts could not form a reductant which had an oxidation-reduction potential more reducing than 0 volt (1,2). For over a decade chloroplasts were known to reduce TPN only photochemically, but recently Tagawa and Arnon (3) have described a system in which chloroplasts reduced TPN also in the dark. The reduction of TPN proceeded in the dark even when the green (chlorophyll-containing) portion of chloroplasts was discarded and only a flavoprotein fraction was retained. The flavoprotein fraction contained the TPN-reducing enzyme of chloroplasts; the other components of the system that reduced TPN in the dark were (a) hydrogen gas and bacterial hydrogenase as the source of reducing power, and (6) a ferredoxin, either from Clostridium pasteurianum (4) or from chloroplasts and photosynthetic bacteria (3), which served as the electron carrier between Hz-hydrogenase and t,he TPN-reducing enzyme. When the flavoprotein ferredoxin-TPN reductase of chloroplasts was recently crystallized (5, 6), its activity was measured photochemically by coupling the enzyme with illuminated grana as the source of reducing power. In the present investigation, which was concerned with the mechanism of action of ferredoxinTPN reductase, the activity of the crystalline enzyme was measured in the dark, by using the Hz-hydrogenase system as a source of reducing power instead of illuminated grana. The advantages of this “dark” method, described herein, over the photochemical method for the study of the mechanism of TPN reduction in chloroplasts are that (a) it eliminates the interference of chloroplast pigments and of act.inic light in the spectrophotometric assay of TPN reduction at 340 rnp, and (b) it dispenses with the “grana” portion of the chloroplasts, thereby making it possible to replace the grana-bound ferredoxin-TPN reductase (6) with the crystalline enzyme, the concentration of which can be varied independently of the photochemically generated reducing power. The results report,ed here show that the mechanism of TPN reduction by ferredoxin-TPX reductase involves first a reduction of the enzyme by reduced ferredosin, followed by a reoxidation of the reduced ferredoxin-TPN reductase by TPN (Fig. 1). The same mechanism was also found to operate in the reduction of DPN by the crystalline enzyme. The previously considered specificity of ferredoxin-TPN reductase for TPN is now explained by the fact that its K, value for TPN is about 0.0025 its K, value for DPN. The relation of these findings to the general problem of TPN reduction by chloroplasts is discussed. A brief account of this work has been published (7).