Ferrous nitrosylated cytochrome c: The unusual strength of the proximal His18-Fe bond

• Published in: Journal of inorganic biochemistry Vol. 247; p. 112338
• Main Authors: De Simone, Giovanna, Monaca, Sara Della, Fattibene, Paola, Bocedi, Alessio, Coletta, Massimo, Ascenzi, Paolo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2023
• Subjects: Cleavage of the proximal His18-Fe bond; Cytochrome c; EPR spectroscopy; Ferrous nitrosylated cytochrome c; Optical absorption spectroscopy; pH effect; Cytochrome c; Optical absorption spectroscopy; pH effect; Ferrous nitrosylated cytochrome c; Cleavage of the proximal His18-Fe bond; EPR spectroscopy
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2023.112338

NO binding to horse heart cytochrome c (hhcyt c) has been investigated as a function of pH by both optical absorption and EPR spectroscopies. Lowering pH from 3.5 to 1.5 induces: (i) a blue-shift of the maximum of the optical absorption spectrum in the Soret region from 415 to about 404 nm, and (ii) the appearance of a strong three hyperfine splitting in the gz region of the EPR spectrum. Both spectroscopic features indicate the cleavage of the proximal His18-Fe(II)-NO bond giving rise to the five-coordinated Fe(II)-NO species. By quantification of the relative weight for the six- and the five-coordinated component in the EPR spectra, the pKa value was determined. The apparent pKa of the proximal His Nε atom (1.8 ± 0.1) is unusually low for a ferrous nitrosylated form since in all investigated ferrous NO-bound heme-proteins the pKa value for the cleavage of the proximal His-Fe(II) bond ranges between 3.7 and 5.8. The pKa value of ferrous nitrosylated hhcyt c indicates that the strength of the proximal His18-Fe(II) bond (= 27.9 kJ/mol) is about 10–22 kJ/mol higher than that observed in all investigated heme-proteins. The strong coordination of the heme-Fe atom by His18 is extremely important to maintain the redox efficiency of cyt c and to keep apoptosis under control. This is a crucial point in tissues, such as retina, where apoptosis might trigger macular degenerative processes.
Lowering pH from 3.5 to 1.5, the cytochrome c(II)-NO solution induces a blue-shift of the maximum of the optical absorption spectrum in the Soret region and the appearance of a 3-lines hyperfine splitting in the gz region of the EPR spectrum. This indicates the cleavage of the proximal His18-Fe(II)-NO bond. [Display omitted]
•Cytochrome c(II)-NO undergoes a reversible spectroscopic transition below pH 3.5.•The pKa value of the reversible protonation of the Nε atom of His18 is 1.8 ± 0.1.•Upon lowering pH, the heme-Fe(II)-NO atom undergoes five coordination.•The energy of the proximal His18-Fe bond of cytochrome c(II)-NO is 27.9 kJ/mol.