Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR.

Abstract

Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an α₂β₂ heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.