Abstract
Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction to form chlorophyllide. Three subunits of DPOR, BchL, BchN and BchB, show significant similarity to those of three nitrogenase subunits, NifH, NifD and NifK, respectively. In the previous study, we have confirmed the nitrogenase-like features of DPOR by the reconstitution of purified components. A crystal structure of NB-protein showed that the [4Fe-4S] cluster is coordinated by three Cys residues of BchN (Cys26, Cys51, Cys112) and Asp residue of BchB (Asp36). To address the functional significance of these residues in the cluster coordination and the Pchlide reduction, we have constructed a series of strep-tagged NB-protein mutants of the Cys and Asp residues (C26A/S, C51A/S, C112A/S, D36C/A/S, C95S/A). The activity of the mutant NB-proteins was evaluated in vivo by the complementation of the bchN- or bchB-disrupted mutants of R. capsulatus and in vitro by biochemical analysis of the purified NB-protein.
