Breakdown of Chlorophyll: Biochemicial Properties of Red Chlorophyll Catabolite Reductase

While the basic structures of chlorophyll catabolites in higher plants are currently elucidated, the mechanism of the enzymatic transformations is still far from being clear. Red chlorophyll catabolite reductase (RCCR) is a key enzyme involved in chlorophyll breakdown in higher plants, which furnishes the fleetingly observable fluorescent chlorophyll catabolites by reduction of enzyme bound red chlorophyll catabolite. RCCR is a representative of the bilin reductases, a remarkable new class of reductases that are important in both tetra-pyrrole catabolism and biosynthesis (of some linear tetrapyrroles). These reductases i) do not need redox-active cofactors and ii) have still unknown means to achieve high enzymatic control of regio- and stereo-chemistry of the reduction. Interest in RCCR is therefore more general since the degradative pathways of the plant chlorophylls and the enzymatic formation of phycobilins from heme appear to share important common features. In this project we are interested in biochemically characterizing the reduction catalyzed by red chlorophyll catabolite reductase (RCCR) by in vitro studies with enzyme preparations isolated from senescent barley as well as produced by overexpression of the gene from Arabidopsis thaliana. RCCR is indicated to not need a low molecular weight cofactor for its` task of reducing the red chlorophyll catabolite. In vivo, red chlorophyll catabolite is still bound to the oxygenase that generates it, and is reduced by RCCR to produce the fluorescent chlorophyll catabolite. We are interested in determining conditions for that remarkable enzymatic reduction catalyzed by RCCR alone. Our planned investigations are therefore geared at (i) finding optimal experimental conditions for the efficient (preparative) in vitro reduction of red chlorophyll catabolite catalyzed by red chlorophyll catabolite reductase from natural isolates and from an E. coli overexpression system (ii) elucidating the biochemical mechanism of the suggested unusual enzymatic reduction by red chlorophyll catabolite reductase and to investigate the factors relevant for achieving the regio- and stereo-selectivity of the enzymatic double bond reduction. This work is planned to contribute primarily to a better understanding of a key enzymatic transformation involved in chlorophyll breakdown in higher plants. The studies may also be more broadly relevant for the class of newly discovered bilin reductases, and the insights gained from these studies could provide useful information for subsequent structural biological studies. A better understanding of chlorophyll breakdown (and of porphyrin metabolism) may contribute to improvements in agriculture and may also be relevant to developments in economics and ecology.