biostudies-literatureJiang TNational Institute of Environmental Health SciencesNIEHS NIH HHSDivision of Molecular and Cellular BiosciencesNational Institute of General Medical SciencesNIGMS NIH HHS1395-1411https://www.ebi.ac.uk/biostudies/studies/S-EPMC11893013biostudies-literatureUnknown63(11)Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., <i>Biochemistry</i>, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodiBiochemistryMapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.PMC118930132041692P20GM130422P30ES032755P30 ES032755P20 GM130422R01 GM133973GM133973Zhang HGyawali YPUnderbakke ESJiang TWan GFeng CfalseMapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., <i>Biochemistry</i>, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodi2024-01-01T00:00:00Z2024 Jun2025-04-22T11:10:01.625Z2025-04-05T23:54:47.661ZS-EPMC118930133874754510.1021/acs.biochem.4c00157