Crossing Enzymatic Boundaries by Coupling BchNB with the Nitrogenase Cofactor Precursor

The dark-operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key step in photosynthetic pigment biosynthesis. Structurally and mechanistically related to nitrogenase, DPOR consists of a reductase (BchL) and a catalytic component (BchNB) homologous to the reductase (NifH) and catalytic component (NifDK) of Mo-nitrogenase. Structural alignment of Rhodobacter capsulatus (Rc) BchNB with Azotobacter vinelandii (Av) NifDK and the cofactor maturase NifEN reveals a conserved α₂β₂ architecture and a shared cofactor-insertion path linking their respective prosthetic-like group/cofactors (Pchlide, M-cluster, L-cluster), suggesting the possibility of generating chimeric proteins with novel reactivities. Herein, Pchlide-free RcBchNB (RcBchNB^apo) is reconstituted with the L-cluster extracted from AvNifEN to yield a hybrid protein (RcBchNB^L) capable of reducing N₂ and C₁ substrates (CN^-, CO) to NH₃ and hydrocarbons, respectively, in the presence of a strong reductant (Eu^II-DTPA). In contrast, reconstituting Pchlide-bound RcBchNB with the L-cluster yields minimal activity, indicating that Pchlide and the L-cluster compete for a common binding site, as supported by Boltz-2 modeling. These findings support the hypothesis of an intertwined evolution of photosynthetic and nitrogen-fixing enzymes and outline a framework for engineering chimeric metalloenzymes that couple light capture with nitrogenase-like catalysis in the future.