Reductive activation of E. coli respiratory nitrate reductase

Area of Science:

• Biochemistry
• Enzymology
• Bioenergetics

Background:

• Mo/W-bisPGD enzymes, including Escherichia coli nitrate reductase A (NarGHI), are crucial for anaerobic respiration.
• Previously, inactive enzyme species in as-prepared samples complicated spectroscopic studies due to indistinguishable signatures.
• NarGHI facilitates anaerobic respiration using nitrate as the terminal electron acceptor.

Purpose of the Study:

• To investigate the functional heterogeneity of purified NarGHI.
• To characterize the activation process of inactive species within NarGHI.
• To correlate electrochemical and spectroscopic data to identify the nature of inactive species.

Main Methods:

• Protein film voltammetry was employed on NarGH films.
• Electrochemical data was correlated with Electron Paramagnetic Resonance (EPR) spectroscopy.
• Analysis focused on Mo(V) signals and FeS clusters near the Mo center.

Main Results:

• Purified NarGHI contains 20-40% inactive species that activate upon initial reduction.
• Activation occurs via a two-step mechanism: a reversible non-redox reaction followed by irreversible reduction.
• Inactive species were not associated with major Mo(V) EPR signals or proximal FeS clusters.
• The study ruled out acid-base equilibrium for major Mo(V) EPR signatures.

Conclusions:

• NarGHI is purified in a functionally heterogeneous state with a significant proportion of initially inactive enzyme.
• The activation mechanism of these inactive species involves distinct redox and non-redox steps.
• This research clarifies the nature of inactive species in NarGHI, improving future spectroscopic investigations of the Mo/W-bisPGD enzyme family.