Cytochrome c'_β-Met Is a Variant in the P460 Superfamily Lacking the Heme-Lysyl Cross-Link: A Peroxidase Mimic...

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Bacterial cytochromes (cyt's) in the P460 family are characterized by a unique heme-protein cross-link involving a lysyl residue.
• This cross-link is crucial for the function of cytochromes P460 in ammonia-oxidizing bacteria (AOB).

Purpose of the Study:

• To identify and characterize novel cytochrome variants within the P460 family.
• To investigate the structural and functional implications of variations in the heme-cross-linking residue.

Main Methods:

• Proteomics analysis of the periplasmic fraction of *Nitrosomonas europaea*.
• Optical spectroscopy to assess heme spin state and cross-linking.
• Mössbauer and electron paramagnetic resonance (EPR) spectroscopy for electronic structure determination.
• Enzyme kinetics assays to determine peroxidase activity.

Main Results:

• Identification of a novel cytochrome, designated cyt c’β-Met, containing a methionyl residue instead of the typical lysyl residue at the cross-linking site.
• Cyt c’β-Met lacks the characteristic heme-protein cross-link and exhibits high-spin (S=5/2) character.
• Cyt c’β-Met binds hydrogen peroxide (H2O2) to form a ferryl (FeIV═O) Compound II-like species and displays guaiacol-dependent peroxidase activity (kcat = 20.0 ± 1.2 s−1).
• In contrast, native cyt P460 shows heme inactivation with H2O2, but mutagenesis of the cross-linking lysine to alanine restores activity.

Conclusions:

• The absence of the heme-protein cross-link in cyt c’β-Met alters its reactivity, enabling peroxidase activity and stable ferryl intermediate formation.
• This finding highlights the structural diversity within the P460 cytochrome family and its functional consequences.
• The study provides insights into the evolution and catalytic mechanisms of bacterial cytochromes.