PARENT SESSION

Symposium S5C Biosynthesis and assembly: Pigments
Wednesday September 1st, 2004 10:20 AM-12:20 PM Room 511D
Chair: Sabeeha Merchant
Co-Chair: Sam Beale

Characterization of a nitrogenase-like enzyme catalyzing protochlorophyllide reduction from Rhodobacter capsulatus. Jiro Nomata¹, Kazuhito Inoue², Masaharu Kitashima², Lee Swem³, Carl Bauer³, Yuichi Fujita^*,1, ¹ Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan² Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, Japan³ Department of Biology, Indiana University, Bloomington, Indiana, USA

ABSTRACT- Most photosynthetic organisms except for angiosperms have the ability to synthesize chlorophyll or bacteriochlorophyll in the light-independent manner. Dark-operative protochlorophyllide oxidoreductase (DPOR) plays a crucial role for the light-independent (bacterio)chlorophyll biosynthesis. However, biochemical properties of DPOR are still largely unknown. Here we report characterization of the two components of DPOR, BchL and BchN-BchB, by the use of cells over-expressing the components in Rhodobacter capsulatus. A pair of plasmids, pYCL10 and pYCNB111, constructed on pJRD215, a broad host range vector, to over-express BchL and BchN-BchB, respectively, under control of the puc promoter was introduced into cells of R. capsulatus. High and stable DPOR activity was reproductively detected by mixing crude extracts of the two transconjugants harboring the two plasmids in anaerobic conditions. Using this assay system, we found that 1) the apparent Km for protochlorophyllide of DPOR is 11 microM, 2) upon exposure to the air, only the activity of BchL component is quickly lost with half life of 30 min in contrast to the stable activity of BchN-BchB component, and 3) ferredoxin from maize functions as an electron donor for DPOR in the absence of dithionite. The elution profiles of the activities of BchL and BchNB in gel filtration chromatography indicated that BchL and BchN-BchB components are a homodimer and a heterotetramer ((BchN)2(BchB)2), respectively. BchL component purified by the use of affinity tag showed a significant EPR signal with a g-value of 1.92, which is characteristic of a [4Fe-4S] cluster. These features are in good agreement with the nitrogenase-like model that we previously proposed. BchL component, especially, has the molecular feature very similar to nitrogenase Fe protein. In contrast, BchN-BchB component has somewhat different properties from nitrogenase MoFe-protein, which seems to be attributed to the great difference between the substrates, protochlorophyllide for DPOR and dinitrogen for nitrogenase.

KEY WORDS: protochlorophyllide reduction, bacteriochlorophyll biosynthesis, nitrogenase, Rhodobacter capsulatus