Photoreduction of Zinc Protopheophorbide b with NADPH-Protochlorophyllide Oxidoreductase from Etiolated Wheat (Triticum aestivum L.)

Abstract

A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…The Pchlide specificity of POR is largely unknown. However, detergenttreated POR enzyme preparations from wheat etioplasts convert 3-monovinyl Pchlide a but not the esterified forms of this compound to Chlide (Schoch et al, 1995), which confirms previous results reported by Griffiths (1980). The wheat enzyme also converts Zn2'-protopheophorbide b, which is the Zn2+ analog of Pchlide b, to Zn'+-pheophorbide (Schoch et al, 1995).…”

“…However, as described above for Pchl a, the esterified forms of Pchlide b (i.e. Pchl b) could not be photoreduced by this enzyme preparation (Schoch et al, 1995).…”

“…Because the functions of most of these different Pchlide forms are largely unknown, one cannot exclude that certain members of the Pchlide family could be the preferred substrates for either PORA or PORB and thus could give rise to distinctive routes of Chlide synthesis. Studies with detergent-solubilized POR enzyme preparations from wheat or barley etioplasts (Schoch et al, 1995) cannot explicitly answer the question on the substrate specificities of PORA and PORB, because such preparations always contain a mixture of both POR enzymes (Holtorf et al, 1995).…”

“…As a first step, a photoactive ternary POR-Pchlide-NADPH complex is formed. Such a complex is spectroscopically detectable in prolamellar bodies of wheat and barley etioplasts (Griffiths, 1975(Griffiths, , 1978Oliver and Griffiths, 1982), in greening barley leaves (Franck and Strzalka, 1992), and can also be reconstituted in vitro with either detergent-solubilized enzymes, such as those from wheat or barley etioplasts (Griffiths, 1978;SanteI and Apel, 1981;Oliver and Griffiths, 1982;Schoch et al, 1995), or with the cDNA-encoded, in-vitro-synthesized PORA and PORB precursor proteins from barley (Holtorf et al, 1995;Reinbothe et al, 1995a,b). Wilks and Timko (1995) proposed that NADPH binds to a region of the polypeptide which is similar to that of short-chain alcohol dehydrogenases (Baker, 1994 dues (Tyr275 and Lys279) were suggested to be part of the active site of the pea enzyme.…”

“…However, detergenttreated POR enzyme preparations from wheat etioplasts convert 3-monovinyl Pchlide a but not the esterified forms of this compound to Chlide (Schoch et al, 1995), which confirms previous results reported by Griffiths (1980). The wheat enzyme also converts Zn2'-protopheophorbide b, which is the Zn2+ analog of Pchlide b, to Zn'+-pheophorbide (Schoch et al, 1995). This finding suggests that POR of wheat etioplasts might exhibit no strict specificity with regard to the 3-monovinyl forms of either Pchlide u or b.…”