Secondary-Sphere Control of Electronic Structure and Ligand Basicity in Mo-Nitrogenase: A QM/MM Analysis Guided by Se K-Edge HERFD XAS

Molybdenum nitrogenase catalyzes biological nitrogen fixation using a complex metallocofactor (FeMoco) containing three μ2-“belt”sulfides that exhibit distinct reactivity during catalysis. In particular, protonation at the S2B site imparts lability that facilitates substrate binding and may enable this sulfide to act as a proton donor. Although FeMoco is inactive outside its protein environment, how secondary-sphere residues tune its geometric and electronic structure to enable function remains unclear. We have previously shown that site-selective Se substitution of the μ2-sulfides provides a direct probe of the ligand environment using Se K-edge high-energy-resolution fluorescence-detected X-ray absorption spectroscopy (HERFD XAS). Despite the cofactor’s approximate C3 symmetry, Se-substituted sites display pronounced differences in Se HERFD XAS pre-edge intensities. Utilizing QM/MM calculations, we show that hydrogen-bonding (H-bonding) residues polarize the S3A and S5A sulfides, reducing metal–ligand covalency and attenuating pre-edge intensity, whereas hydrophobic residues surrounding S2B localize electron density and enhance its pre-edge intensity. These results demonstrate how hydrophobicity and H-bonding within the secondary coordination sphere modulate FeMoco electronic structure. More broadly, they show how the protein environment tunes ligand basicity to favor selective S2B protonation and provide design principles for controlling enzymatic function.