Structural Characterization of Cytochrome from an Ammonia-Oxidizing Bacterium

Area of Science:

• Biochemistry and Structural Biology
• Microbial Metabolism
• Enzyme Structure-Function Relationships

Background:

• Nitrosomonas europaea possesses two P460 superfamily cytochromes with predicted structural similarities.
• Cytochrome P460 catalyzes hydroxylamine oxidation via a heme-lysyl cross-link, producing nitric and nitrous oxides.
• The second cytochrome, $$, lacks this cross-link and interacts with H₂O₂, forming a ferryl species.

Purpose of the Study:

• To determine the 1.80 Å crystal structure of cytochrome $$ from N. europaea.
• To elucidate the structural basis for its distinct biochemical properties compared to cytochrome P460 and related enzymes.
• To understand the functional implications of active site variations and the absence of the heme-lysyl cross-link.

Main Methods:

• X-ray crystallography to obtain high-resolution structural data (1.80 Å).
• Comparative structural analysis with homologous cytochromes, including P460 and cytochrome cbb₃.
• Bioinformatic analysis of active site residues and potential proton transfer pathways.

Main Results:

• The crystal structure of cytochrome $$ reveals a characteristic P460 β-sheet fold with unique features, including a novel lasso-like loop.
• The active site differs from its homolog, cytochrome cbb₃, notably with a distal arginine instead of phenylalanine, and lacks a critical glutamate for hydroxylamine oxidation.
• The structure explains the absence of the methionyl cross-link, although its potential formation under specific conditions is not entirely ruled out.

Conclusions:

• The determined structure of cytochrome $$ highlights significant structural divergence from other P460 members, particularly in the active site.
• The absence of key residues explains the inability of cytochrome $$ to perform hydroxylamine oxidation.
• The structural findings provide insights into the functional specialization of P460 superfamily cytochromes and their roles in microbial metabolism.