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 α 2 β 2 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 Rc BchNB ( Rc BchNB apo ) is reconstituted with the L‐cluster extracted from Av NifEN to yield a hybrid protein ( Rc BchNB L ) capable of reducing N 2 and C 1 substrates (CN − , CO) to NH 3 and hydrocarbons, respectively, in the presence of a strong reductant (Eu II ‐DTPA). In contrast, reconstituting Pchlide‐bound Rc BchNB 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.