Abstact

Nitrosospira multiformis is a soil ammonia-oxidizing bacterium (AOB) that fixes CO(2) via RubisCO using energy derived from ammonia oxidation. Despite its ecological and agricultural relevance, the structure of N. multiformis RubisCO (NmRubisCO) remained unknown. Here, we report the cryo-EM structure of NmRubisCO at 2.26 A resolution, determined through single-particle analysis of protein copurified with the membrane fraction following cell lysis and ultracentrifugation without column chromatography. Analysis revealed an L(8)S(8) hexadecameric Form IC (red-like) RubisCO with a six-residue insertion in the betaB-betaC loop (BC loop) of the large-subunit N-terminal domain that forms a solvent-exposed, well-ordered protrusion. The active site lacks carbamylation at K206 and Mg(2+) binding, representing an inactive state. A putative metal ion coordinated by H296, H298, and H331, tentatively assigned as Zn(2+) based on coordination geometry and distances, was identified near the catalytic center; this His-coordinated metal site has not been described in other RubisCO structures. Loop 6 adopts a closed-like conformation in the absence of substrate, a configuration consistent with a potential requirement for activase-mediated remodeling. The C-terminal extension of the small subunit forms interdimer beta-sheet interactions that stabilize the holoenzyme. These structural features provide a framework for understanding Form IC RubisCO in carboxysome-less soil AOB.