Spin‐equilibrium and heme‐ligand alteration in a high‐potential monoheme cytochrome (cytochrome c₅₅₄) from Achromobacter cycloclastes, a denitrifying organism

A c‐type monoheme cytochrome c₅₅₄ (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme‐methionine coordination (low‐spin form) coexisting with a minor high‐spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high‐spin form at all pH values, attaining a minimum at intermediate pH values. The mid‐point redox potential determined by visible spectroscopy at pH 7.2 is + 150 mV. The pH‐dependent spin equilibrum and other relevant structural features were studied by 300‐MHz ¹H‐NMR spectroscopy. In the oxidized form, the ¹H‐NMR spectrum shows pH dependence with pK_a values at 5.0 and 8.9. According to these pK_a values, three forms designated as I, II and III can be attributed to cytochrome c₅₅₄. Forms I and II predominate at low pH values, and the ¹H‐NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low‐field region of the NMR spectra. Above pH 9.6, form III predominates and the ¹H‐NMR spectrum is characterized by down‐field hyperfine‐shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at ≃ 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH‐dependent features of the ¹H‐NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti‐Curie temperature dependence. In the ferrous state, the ¹H‐NMR spectrum shows a methyl proton resonance at −3.9 ppm characteristic of methionine axial ligation. The electron‐transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 × 10⁴ M⁻¹ s⁻¹ at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at g_max≃ 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.