Decoding in-cell respiratory enzyme dynamics by label-free in situ electrochemistry

Area of Science:

• Biochemistry
• Microbiology
• Bioenergetics

Background:

• Studying enzyme kinetics in living cells is challenging due to limitations of traditional in vitro assays.
• Existing methods often isolate enzymes, failing to capture complex cellular interactions.

Purpose of the Study:

• To develop and validate a novel whole-cell electrochemical assay for in vivo enzyme kinetics.
• To investigate the Michaelis-Menten kinetics of respiratory enzymes within their native cellular environment.

Main Methods:

• Utilized a whole-cell electrochemical assay to control microbial current generation.
• Determined in vivo kinetic parameters (Km, Ki, k) for periplasmic nitrite (NrfA) and fumarate (FccA) reductases.
• Employed a mutant strain lacking CymA to assess its regulatory role.

Main Results:

• Successfully extracted in vivo kinetic parameters for NrfA and FccA.
• Observed that NrfA kinetics in vivo matched purified enzyme kinetics, while FccA showed distinct behavior.
• Demonstrated CymA's significant role in modulating FccA kinetics, contradicting solely molecular crowding explanations.

Conclusions:

• The developed whole-cell electrochemical assay provides a powerful platform for studying cellular respiratory enzyme kinetics.
• Cellular factors, such as hub proteins like CymA, play a critical role in enzyme kinetics beyond molecular crowding.
• This research opens new avenues for understanding bioenergetics and developing medical applications.