Spectroscopic and theoretical study of a stable iron(III)-hyponitrite complex in chlorite dismutase

Nitric oxide (NO) reduction to nitrous oxide (N2O) is a key step in bacterial denitrification, widely proposed to proceed via a hyponitrite (HN) intermediate bound to iron(III) metal cofactors, yet to date, there is no structurally defined iron-HN complex in any hemoprotein and no detailed spectroscopic characterization of such a complex. Here, we present direct spectroscopic and computational evidence for the formation of a stable HN adduct in ferric Dechloromonas aromatica chlorite dismutase (DaCld). UVvis titrations show that HN binds DaCld with a dissociation constant (K D ) of 75.2 μM, about ten-fold lower than nitrite, even though nitrite more closely mimics the native substrate, chlorite. Both adducts exist as six-coordinate thermal spin-state equilibrium at room temperature, converting to pure low-spin species at low temperatures. Stopped-flow kinetics yield association (k on ) and dissociation (k off ) rate constants for HN of 315 mM -1 s -1 and 10 s -1 , respectively. Electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman (RR) spectroscopies show that the HN adduct adopts an O-bound coordination but have so far failed to provide information on the adduct's protonation state. QM/MM calculations support multiple plausible binding geometries at the Fe(III) open coordination site, each requiring proton transfer from the distal Arg183 to generate a monoprotonated HONNO- species. Furthermore, calculations demonstrate that an O-bound cis-HN complex spontaneously yields N 2 O and a ferric-hydroxide species. These findings provide further experimental and theoretical context for stable and reactive iron-HN complexes in biological systems.