Proton and electron pathways in the bacterial nitric oxide reductase

Area of Science:

• Biochemistry
• Enzymology
• Bioenergetics

Background:

• Bacterial nitric oxide reductase (NOR) is crucial for nitrogen cycling and microbial respiration.
• Understanding the electron and proton transfer mechanisms in NOR is key to elucidating its catalytic function.
• Previous studies suggest similarities between NOR and heme-copper oxidases, but detailed mechanistic steps remain unclear.

Purpose of the Study:

• To investigate the electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) using combined optical spectroscopy and electrometry.
• To elucidate the pathway and kinetics of intermediates during nitric oxide reduction.
• To compare the electron transfer topology in NOR with that of heme-copper oxidases.

Main Methods:

• Optical spectroscopy to monitor changes in heme redox states and ligand binding.
• Electrometry to measure electric potential generation across liposome-embedded NOR during turnover.
• Kinetic analysis of spectral and electrical signals to determine rate constants for intermediate steps.

Main Results:

• NOR turnover in liposomes is electroneutral, indicating protons and electrons are transferred from the same membrane side.
• Electron transfer pathway mirrors heme-copper oxidases: heme c -> heme b -> binuclear site (heme b3/FeB).
• Identified distinct intermediate states during NO reduction with specific rate constants, including a ferrous NO adduct of heme b3 and a charge separation phase.

Conclusions:

• The electron transfer topology in NOR is comparable to heme-copper cytochrome oxidases.
• Mechanistic models favor a trans mechanism over a cis model involving FeB for NO reduction.
• NOR may utilize a P450-type mechanism where heme b3 is the sole NO binding site during turnover.