Expression, purification and biochemical characterization of the periplasmic nitrate reductase NapA from...

Magnetospirillum gryphiswaldense MSR-1 is a model magnetotactic bacterium whose magnetosome biomineralization is closely linked to intracellular redox regulation. Although the periplasmic nitrate reductase NapA plays an essential role in denitrification and redox control, its biochemical properties in MSR-1 have remained unexplored. In this study, we constructed a heterologous co-expression system for MSR-1 NapA together with its cognate chaperone NapD, successfully producing soluble and biologically active recombinant NapA. The purified enzyme exhibited a characteristic iron-sulfur absorption band near 400 nm, and metal content analysis confirmed the presence of both iron and molybdenum cofactors. Electron paramagnetic resonance spectroscopy further demonstrated the presence of a redox-active [4Fe-4S] cluster and a Mo(V) center, indicating correct cofactor assembly. Enzymatic assays showed that MSR-1 NapA follows typical Michaelis-Menten kinetics toward nitrate, with optimal activity near neutral pH and moderate temperatures. These results establish MSR-1 NapA as a functionally active and structurally stable molybdoenzyme. Collectively, this work provides the first comprehensive biochemical characterization of NapA from a magnetotactic bacterium and offers a solid molecular foundation for understanding how nitrogen metabolism is coupled to redox regulation and magnetosome biomineralization in M. gryphiswaldense MSR-1.