Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Cytochrome bd oxidases are essential bacterial terminal oxidases, particularly under low oxygen conditions.
• These enzymes are vital for the survival of numerous pathogens, making them potential antimicrobial drug targets.
• The largest subunit, CydA, harbors three key redox cofactors: heme b558, heme b595, and the active site heme d.

Purpose of the Study:

• To investigate the oxygen (O2) reduction mechanism in Escherichia coli cytochrome bd-I.
• To elucidate the kinetics and pathway of proton transfer during the catalytic cycle.
• To identify the role of specific subunits and residues in proton transfer.

Main Methods:

• Utilized the flow-flash technique to study rapid enzymatic reactions.
• Employed site-directed mutagenesis to alter specific amino acid residues in the CydB subunit.
• Analyzed pH-dependent kinetics of the peroxy (P) to ferryl (F) transition.

Main Results:

• The P➔F transition rate is pH-dependent, with maximal activity around 10^4 s^-1, decreasing at higher pH.
• This pH dependence suggests rate limitation by an internal proton transfer from a residue with a pKa of approximately 9.7.
• Mutagenesis of Asp58B and Asp105B in CydB significantly impacted catalytic turnover, implicating them in proton transfer. Specifically, Asp105B is crucial for the P➔F transition.

Conclusions:

• A specific proton transfer pathway involving CydB, particularly Asp105B, is critical for the function of E. coli cytochrome bd-I.
• The study identifies a high pKa residue involved in proton transfer and discusses its conservation within the cytochrome bd superfamily.
• Understanding this mechanism provides insights into bacterial respiration and potential targets for antimicrobial development.