Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460

Area of Science:

• Biochemistry and enzymology
• Microbial metabolism and nitrogen cycling

Background:

• Ammonia oxidation by ammonia-oxidizing bacteria (AOB) is vital for nitrification.
• Metalloenzymes like hydroxylamine oxidoreductase (HAO) and cytochrome P460 (cyt P460) control product selectivity (nitric oxide vs. nitrous oxide) during hydroxylamine (NH2OH) oxidation.
• Cyt P460 utilizes a heme P460 cofactor, and structural studies suggest a capping phenylalanine residue influences its activity.

Purpose of the Study:

• To investigate the role of the capping phenylalanine residue in Nitrosomonas europaea cyt P460.
• To determine how this residue affects cofactor maturation and product selectivity during NH2OH oxidation.

Main Methods:

• Site-directed mutagenesis was used to substitute the capping phenylalanine residue in cyt P460.
• Enzyme activity assays were performed to assess NH2OH oxidation under aerobic conditions.
• Kinetic analyses were conducted to evaluate N2O formation and NO binding affinity.

Main Results:

• The bulky phenyl side-chain of the capping phenylalanine promotes the heme-lysine cross-link formation essential for cofactor maturation.
• Mutations in the capping phenylalanine residue significantly alter product selectivity, favoring either N2O or NO production.
• The steric and electrostatic properties of the capping phenylalanine side-chain modulate the kinetics of N2O formation and NO binding.

Conclusions:

• The outer coordination sphere of cyt P460, specifically the capping phenylalanine, is critical for both enzyme activity and product specificity.
• This residue plays a dual role in facilitating autocatalytic cofactor maturation and fine-tuning the oxidation pathway of NH2OH.
• Understanding these mechanisms provides insight into the regulation of nitrogen cycling by AOB.