Reconstitution of light-independent protochlorophyllide reductase from purified Bch1 and BchN-BchB subunits - In vitro confirmation of nitrogenase-like features of a bacteriochlorophyll biosynthesis enzyme

Protochlorophyllide reductase catalyzes the reductive formation of chloroph yllide from protochlorophyllide during biosynthesis of chlorophylls and bac teriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photo synthetic bacteria to green (form chlorophyll) in the dark. Genetic and seq uence analyses have indicated that dark proto chlorophyllide reductase cons ists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive format ion of ammonia from dinitrogen. However, unlike the well characterized feat ures of nitrogenase, there has been no previous biochemical characterizatio n of dark protochlorophyllide reductase, In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R, capsulatus as S tag fusion proteins t hat facilitated affinity purification. The third subunit (BchB) was co-puri fied with the BchN protein indicating that BchN and BchB proteins form a ti ght complex. Dark protochlorophyllide reductase activity was shown to be de pendent on the presence of all three subunits, ATP, and the reductant dithi onite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed.