Proton transfer in bacterial nitric oxide reductase

Understanding how protons move during enzymatic reactions is a central challenge in biochemistry. While oxygen-reducing enzymes are well-studied, less is known about how protons are transferred in NO-reducing enzymes. Prior research has shown that oxygen-reducing enzymes use proton-coupled electron transfer to generate a proton gradient. However, the proton pathway in NO reductase remains unclear. This uncertainty has limited progress in understanding the coupling of electron and proton transfer in this enzyme. No prior work had resolved the specific proton donor or pathway in this system. The lack of structural and functional data has left a gap in the field. Researchers have proposed that proton transfer is essential for NO reduction, but the mechanism remains speculative. This gap motivated a closer look at the proton-coupled reactions in NO reductase. The need to identify the proton pathway is driven by its role in enzyme function.

This study aimed to investigate the proton transfer mechanism in bacterial nitric oxide reductase. The researchers focused on identifying the proton donor and transfer pathway during NO reduction. They sought to determine how protons are coupled with electron transfer in this enzyme. The study used a chemically less reactive oxidant, O2, to probe the proton transfer process. By analyzing the reaction between fully reduced NOR and O2, the team aimed to uncover the proton donor's identity and location. The goal was to link the observed proton transfer to the enzyme's active site. The researchers also aimed to propose a structural model for the proton pathway. Their findings could clarify how protons are transported in this unique class of enzymes.

The study utilized a combination of biochemical and structural approaches to investigate proton transfer in NOR. The researchers analyzed the reaction between fully reduced NOR and O2 as a model system. They measured the rate of proton-coupled electron transfer in this reaction. The pKa of the proton donor was determined using kinetic methods. The team used a structural model of NOR to propose candidate residues for the proton donor. They compared the reaction kinetics with and without proton sources to infer the donor's role. The study also examined the spatial arrangement of residues near the catalytic site. The researchers tested whether the proton donor is conserved across related enzymes.

The reaction between fully reduced NOR and O2 revealed a proton-coupled electron transfer mechanism. The proton transfer was rate-limited by a group with a pKa of 6.6. This group is likely an amino acid residue near the active site of NOR. The proton donor is proposed to function during both O2 and NO reduction. The study identified potential candidates for the proton donor in the structural model. The proton transfer pathway was hypothesized to connect the periplasmic side to the active site. The findings suggest that the proton donor is conserved in related enzymes. The results support a model in which proton transfer is coupled to electron transfer in NOR.

The study provides evidence for a proton donor with a pKa of 6.6 in NOR. This donor is likely an amino acid residue near the active site. The proton transfer pathway is proposed to connect the periplasmic side to the catalytic site. The findings support a model of proton-coupled electron transfer in NO reduction. The researchers suggest that the proton donor is conserved in related enzymes. The study does not claim that this proton donor is the only contributor to the reaction. The results are consistent with a structural model of NOR. The authors propose that this proton donor may also function in O2 reduction.