Extensive study of the electronic structure of Fe-NO complexes using a variety of spectroscopic methods was attempted to understand how iron controls the binding and release of nitric oxide. The comparable energy levels of NO p* orbitals and Fe 3d orbitals complicate the bonding interaction within Fe À NO complexes and puzzle the quantitative assignment of NO oxidation state. Enemark-Feltham notation, {Fe(NO) x } n , was devised to circumvent this puzzle. This 40-year puzzle is revisited using valence-to-core X-ray emission spectroscopy (V2C XES) in combination with computational study. DFT calculation establishes a linear relationship between DE s2s*-s2p of NO and its oxidation state. V2C Fe XES study of Fe À NO complexes reveals the DE s2s*-s2p of NO derived from NO s 2s */s 2p !Fe 1s transitions and determines NO oxidation state in Fe À NO complexes. Quantitative assignment of NO oxidation state will correlate the feasible redox process of nitric oxide and Fe-nitrosylation biology.After being discovered as the endothelium-derived relaxing factor (EDRF) and named as molecule of the year in 1992, nitric oxide was reported to trigger versatile signal transduction pathways through Fe-nitrosylation, S-nitrosation of thiol-containing proteins, Tyr-nitration, and N-nitrosation.[1]Among these signal transduction pathways, Fe-nitrosylation features a diverse set of physiological processes and various reaction mechanisms ascribed to alternative Fe active-site structure and feasible redox propensity of nitric oxide. Interplay between the NO and Fe center in different proteins modulates vascular relaxation, transcriptional activation, enzymatic function in Krebs cycle, and iron homeostasis. [1d, 2] Formation of mononitrosyl iron complex (MNIC) through rapid binding of nitric oxide toward the regulatory ferrous heme group of soluble guanylate cyclase (sGC) accelerates the formation of cGMP and activates the vascular relaxation of blood vessels.[1d] Investigation of the biological function of Fe-nitrosylation led to the discovery of dinitrosyl iron complexes (DNICs) during nitrosylation of [Fe-S] proteins, chelatable iron pool (CIP), and ferritin. [2b, 3] As a result of nitrosylation, DNICs are one of the major forms for storage of nitric oxide and transnitrosation. Endogenous formation of DNICs derived from nitrosylation of CIP triggers the subsequent transnitrosation to afford cellular S-nitrosothiol.[3a] Induction of NO-dependent upregulation of celluar heat shock protein 70 and in vivo protein S-nitrosylation by treatment of exogenous DNICs was also reported. [4] Extensive study of the electronic structure of nitrosyl iron complexes using a variety of spectroscopic methods was attempted to understand how the metal iron utilizes its intrinsic reactivity and redox propensity for the stabilization of nitric oxide in the form of FeÀNO complexes.[5] These FeÀ NO complexes, in the meantime, are ready to release nitric oxide after accomplishment of NO-related signaling process or encountering with the NO-respons...