Substrate sensing institutes sequential and asymmetric electron transfer in the nitrogenase-like DPOR Complex

Abstract

Dark-operative protochlorophyllide oxidoreductase (DPOR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key penultimate step in the biosynthesis of bacteriochlorophyll. DPOR shares structural homology with nitrogenase and is made of electron donor (BchL) and electron acceptor (BchNB) component proteins. ATP driven assembly of the BchL and BchNB proteins drives electron transfer and Pchlide reduction. BchNB is composed of two subunits each of BchN and BchB arranged as an 2{beta}2 heterotetramer. Here, we describe extensive allosteric communication between the two identical active sites in BchNB that drives sequential and asymmetric electron transfer. Pchlide binding and electron transfer activities in one half of the BchNB tetramer allosterically regulates activities in the other half. Pchlide binding is sensed and recognized in trans by an Asp274 from the opposing half and is positioned in the active site to likely serve as the initial proton donor. An Asp274 to Ala substituted DPOR binds to two Pchlide molecules in the BchNB complex but is unable to conformationally poise one Pchlide molecule. Thus, stalling Pchlide reduction in both active sites. The [4Fe-4S] cluster of the BchNB protein is pre-reduced and donates the first electron to Pchlide, a mechanism similar to the deficit-spending model observed in nitrogenase. In half-reactive DPOR complexes, incapacitating proton donation in one half generates a stalled intermediate and Pchlide reduction in both halves is abolished. The results showcase long-range allosteric communication and sequential ET in the two symmetric halves. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.