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

Abstract

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

Figure 1

Heme-nitrite linkage isomers.

Figure 2

Effect of nitrite on the UV-visible absorption spectrum of Fe(III) L16G AXCP.

Figure 3

Midfrequency RR region of Fe(III)-nitrite L16G AXCP showing the identification of δ(NO₂) and ν_s(NO₂) vibrations via substitution with ¹⁵N¹⁸O₂⁻.

Figure 4

Low frequency RR spectrum of Fe(III)-nitrite L16G AXCP. Inset shows the identification of the ν(Fe–NO₂) vibration via isotopic substitution. Isotope data for the ∼400–800 cm⁻¹ region is shown in Figure S7).

Figure 5

Room-temperature FTIR difference spectra obtained after illumination of L16G Fe(III)-nitrite complexes with 351 nm.

Figure 6

Optimized structures of the L16G AXCP Fe(III)-nitrite complex showing (A) nitro and (B) nitrito coordination modes. Note: His120, Gly16, and Met19 residues are truncated at Cα carbons as CH₃ groups.

Figure 7

Low-dose (0.16 MGy) X-ray crystal structure of the heme environment of L16G-nitrite AXCP at 1.06 Å resolution showing a weighted electron density F_o-F_c omit map (green) superimposed on the 2F_o-F_c map (gray) contoured at 0.44 e/ų. A mixture of nitrite and water were modeled as heme ligands in the proportion 0.25:0.75 to account for the ligand electron density with B-factors 19.6/17.8/19.7 Ų for nitrite O/N/O atoms, 10.4 Ų for water, and 8.9 Ų for the Fe atom. The Fe–N(nitrite) distance is 2.1 Å, Fe–N–O angles 118°/120°, and Fe-water distance 2.07 Å.