Interdomain hydride transfer in proton-translocating transhydrogenase

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Kinetics

Background:

• Transhydrogenase is a key enzyme in cellular energy metabolism, facilitating hydride and proton transfer.
• Understanding the mechanism of transhydrogenase is crucial for elucidating cellular bioenergetics.

Purpose of the Study:

• To investigate the structural, functional, and dynamic aspects of transhydrogenase, focusing on hydride transfer and proton translocation.
• To explore the role of individual domains and specific regions in the enzyme's catalytic activity.

Main Methods:

• Utilized recombinant nucleotide-binding domains (I and III) of transhydrogenase.
• Performed transient state kinetics experiments to analyze reaction dynamics.
• Employed NMR spectroscopy and site-directed mutagenesis to study a mobile loop region in domain I.

Main Results:

• Hydride transfer was observed to be extremely rapid in the recombinant domain I:III complex, independent of domain II.
• Proton translocation through domain II appears coupled to altered nucleotide binding in domain III.
• A mobile loop in domain I was identified as critical for hydride transfer, interacting with NAD+/NADH.

Conclusions:

• The study provides insights into the modular mechanism of transhydrogenase, separating hydride transfer and proton translocation functions.
• The findings suggest a model where domain II-mediated proton translocation influences nucleotide binding in domain III.
• The mobile loop in domain I plays a significant role in the catalytic efficiency of hydride transfer.