Distinguishing Nitro vs Nitrito Coordination in Cytochrome c′ Using Vibrational Spectroscopy and Density Functional Theory

• DOI: 10.1021/acs.inorgchem.7b01945
• Publication Date: 2017-10-23T15:00:50Z
• Authors: Zach N. Nilsson; Brian L. Mandella; Kakali Sen; Demet Kekilli; Michael A. Hough; Pierre Moënne-Loccoz; Richard W. Strange; Colin R. Andrew
• Keywords: Resonance Raman spectroscopy

Nitrite coordination to heme cofactors is a key step in the anaerobic production of signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has potential coordinate via N- (nitro) or O- (nitrito) atoms manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with influence surrounding protein, therefore particular interest. In this study, we probed Fe(III) heme-nitrite Alcaligenes xylosoxidans cytochrome c′ (AXCP), an NO carrier excludes anions native state but readily binds (Kd ∼ 0.5 mM) following distal Leu16 → Gly mutation remove steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe–NO2), δ(ONO), and νs(NO2) ligand vibrations solution. Illumination 351 UV light results photoconversion {FeNO}6 {FeNO}7 states, enabling FTIR measurements distinguish νas(NO2) from differential spectra. Density functional theory calculations highlight connections between environment, mode, vibrational properties confirm L16G AXCP exclusively through N atom. Efforts obtain complex crystal structure were hampered by photochemistry X-ray beam. Although low dose structures could be modeled mixed (nitro)/H2O population, their photosensitivity partial occupancy underscores value approach. Overall, study sheds on determinants binding provides benchmarks for future studies protein reactions.