Hybrid NMR Reveals Loop-Modulated Substrate Recognition in Multicopper Oxidases

Decoupling substrate binding from catalysis remains a central challenge in mechanistic enzymology, particularly for metalloenzymes in which metal cofactors intrinsically couple molecular recognition to electron transfer. Here, we introduce a hybrid nuclear magnetic resonance (NMR) strategy that isolates binding thermodynamics by exploiting metal-free (apo) variants of multicopper oxidases that abolish turnover while retaining structural integrity and binding competence. By combining ligand-based saturation transfer difference (STD) NMR with protein-centered methionine-selective methyl NMR, we resolve substrate-binding interactions at atomic resolution and directly interrogate how dynamic structural elements shape molecular recognition independently of catalysis. Using the metallo-oxidase McoA, its evolved laccase-like variant 2F4, and their Met-looptruncated counterparts, ligand-centered NMR reveals a conserved aromatic binding epitope for the reporter substrate ABTS across all enzyme backgrounds, while distinguishing specific contacts from transient, nonproductive encounters. Quantitative analysis shows that 2F4 binds ABTS more weakly than wild-type McoA despite exhibiting higher catalytic activity, indicating that directed evolution enhances turnover rather than binding. Methionine-selective methyl NMR identifies substrate-induced perturbations in the Met-loop and adjacent surface residues consistent with heterogeneous, multi-site binding. NMR-guided filtering of ensemble docking models supports two adjacent substrate-access cavities that accommodate multiple transient ligand orientations, validating mechanistic features previously inferred only 2 computationally. Collectively, these results define substrate recognition in multicopper oxidases as tunable, with the Met-loop modulating accessibility, promiscuity, and conformational sampling rather than dictating catalytic chemistry. By decoupling binding from catalysis and quantifying loop-modulated interactions, this hybrid NMR framework provides a generalizable strategy for dissecting substrate-recognition mechanisms in multicopper oxidases and other loop-regulated metalloenzymes.