Journal of Biological Chemistry

Mammalian nitric oxide synthases (NOSs) are flavo-hemoproteins that rely on dynamic interdomain interactions for activity. Calmodulin (CaM) facilitates specific, interdomain FMN–heme interactions that enable inter-subunit FMN–heme electron transfer essential for nitric oxide biosynthesis. Our quantitative cross-linking mass spectrometry (qXL MS) results demonstrate that the abundance of intersubunit cross-links correlates with CaM-induced formation of the docked FMN/heme complex in rat neuronal NOS [Jiang et al., Biochemistry, 2024, 63, 1395–1411]. Here, we extend this methodology to the human inducible NOS (iNOS) isoform, comparing wild-type (wt) and E546N mutant oxygenase/FMN (oxyFMN) constructs under near-native conditions. Using parallel reaction monitoring−based qXL MS, we assessed mutation-induced changes in interdomain dynamics. The E546N mutation substantially reduced abundance of specific intersubunit cross-links between the FMN and heme domains. Cross-links between CaM and iNOS domains were also altered by the mutation, indicating that the changes at the FMN–heme docking interface propagate allosterically throughout the iNOS−CaM complex. Although standard AlphaFold2 structural modeling yielded similar docked architectures for wt and mutant, cross-link-guided AlphaLink2 modeling revealed distinct structural differences. AlphaFold2 subsampling further predicted alternative conformations; consistent with qXL MS data, E546N mutant exhibited a broader distribution of predicted conformations, with an apparent shift toward higher population of undocked states, compared to wt. Importantly, 90% of cross-links were consistent with an ensemble of representative conformations derived from AlphaFold2 subsampling and AlphaLink2 modeling, capturing both docked and undocked states alongside multiple orientations. This integrative qXL MS and AlphaFold2 subsampling strategy provides a quantitative framework for mapping mutation-induced alterations in functional dynamics of NOSs and multidomain proteins in general.

nitric oxide synthase

calmodulin

functional protein dynamics

multidomain protein

electron transfer

flavoprotein

heme

crosslinking mass spectrometry

quantitative crosslinking mass spectrometry

AlphaFold2

subsampling

conformational ensemble

conformations

timsTOF

parallel reaction monitoring

data-dependent acquisition

disuccinimidyl dibutyric urea

electron paramagnetic resonance

hydrogen-deuterium exchange mass spectrometry

interdomain electron transfer

multiple sequence alignment

bi-domain oxygenase/FMN construct of NOS

parallel accumulation-serial fragmentation

predicted local distance difference test

trapped ion-mobility spectrometry

parallel reaction monitoring

predicted template modeling

quantitative cross-linking mass spectrometry

cross-linking mass spectrometry

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé