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bioRxiv · 10.1101/2025.01.21.634024

Molecular sorting of nitrogenase catalytic cofactors

Salinero-Lanzarote, A.·Lian, J.·Namkoong, G.·Suess, D. L. M.·Rubio, L. M.·Dean, D. R.·Perez-Gonzalez, A.
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Abstract

The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that contain the incorrect cofactor type cannot reduce N2 to support diazotrophic growth there must be a way to prevent misincorporation of an incorrect cofactor when different nitrogenase isozyme systems are produced at the same time. Here, we show that fidelity of the Fe-only nitrogenase is preserved by blocking the misincorporation of either FeMo-cofactor or FeV-cofactor during its maturation. This protection is accomplished by a two-domain protein, designated AnfO. It is shown that the N-terminal domain of AnfO binds to an immature form of the Fe-only nitrogenase and the C-terminal domain, tethered to the N-terminal domain by a flexible linker, has the capacity to capture FeMo- and FeV-cofactor. AnfO does not prevent the normal activation of Fe-only nitrogenase because completion of FeFe-cofactor assembly occurs within its catalytic partner and, therefore, is never available for capture by AnfO. These results support a post-translational mechanism involving the molecular sorting of structurally similar metallocofactors that involve both protein-protein interactions and metallocofactor binding while exploiting differential pathways for nitrogenase associated catalytic cofactor assembly.

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Salinero-Lanzarote, A., Lian, J., Namkoong, G., Suess, D. L. M., Rubio, L. M., Dean, D. R., Perez-Gonzalez, A.. 2025-01-21. Molecular sorting of nitrogenase catalytic cofactors. https://doi.org/10.1101/2025.01.21.634024

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