Cryo-EM structure of RubisCO from Nitrosospira multiformis

Nitrosospira multiformis is a soil ammonia-oxidizing bacterium (AOB) that fixes CO₂ 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 Å resolution, determined through single-particle analysis of protein copurified with the membrane fraction following cell lysis and ultracentrifugation without column chromatography. Analysis revealed an L₈S₈ hexadecameric Form IC (red-like) RubisCO with a six-residue insertion in the βB-βC 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 Mg²⁺ binding, representing an inactive state. A putative metal ion coordinated by H296, H298, and H331, tentatively assigned as Zn²⁺ 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 β-sheet interactions that stabilize the holoenzyme. These structural features provide a framework for understanding Form IC RubisCO in carboxysome-less soil AOB.