Photosynthetic pyridine nucleotide reductase. I. Partial purification and properties of the enzyme from spinach.

In 1939, Hill (1) reported the fundamental discovery that isolated chloroplasts possess the ability to affect a portion of the over-all reaction of photosynthesis; namely, the photolysis of water. In the original experiments of Hill, ferric oxalate was used as the hydrogen acceptor. During the past 20 years, a variety of substances has been found to function as oxidant for the Hill reaction (2). Of special interest are the pyridine nucleotides in view of their well known physiological function as coenzymes in numerous enzyme-catalyzed reactions. The ability of illuminated chloroplasts to reduce pyridine nucleotides was first demonstrated in 1951-52 by Vishniac and Ochoa (3, 4), Tolmach (5), and Arnon (6). In these experiments, the formation of reduced pyridine nucleotides was demonstrated indirectly by coupling the photochemical reaction with a suitable dehydrogenase and measuring the formation of the product of the dehydrogenase system. No directly measurable reduction of pyridine nucleotides was observed in the absence of the coupling system (7). It was, therefore, suggested (4, 7) that the inability of pyridine nucleotides to undergo directly measurable reduction was a consequence of their low oxidation-reduction potential (Eo’ at pH 7 = -0.32 volt) since most substances that are effective as oxidants in the Hill reaction have high oxidation-reduction potentials (Eo1 at pH 7 = +O.l to +0.4 volt). In 1956, San Pietro and Lang (8) presented evidence that, under the proper conditions, photochemical reduction of pyridine nucleotides can indeed be demonstrated directly. It was shown that when DPW is