Nitrite binding modes in ferric heme proteins probed by HYSCORE spectroscopy

Area of Science:

• Bioinorganic Chemistry
• Biophysics
• Enzymology

Background:

• Nitrite is vital in nitrogen cycling and biochemical reactions, often interacting with heme proteins.
• Understanding nitrite's coordination to heme iron is key, with known N-nitro and O-nitrito binding modes.
• Previous work suggested mixed ligation modes in chlorite dismutase (Cld).

Purpose of the Study:

• To investigate and differentiate nitrite ligation modes (N-nitro vs. O-nitrito) in heme systems.
• To establish a reliable spectroscopic tool for analyzing nitrite coordination chemistry.
• To provide direct evidence for O-nitrito binding in specific heme proteins.

Main Methods:

• Utilized hyperfine sublevel correlation (HYSCORE) spectroscopy on NO2-ligated ferric myoglobin and Cld enzymes.
• Employed 15N-labeled nitrite to distinguish its signals from endogenous nitrogen nuclei.
• Applied Electron Paramagnetic Resonance (EPR) and crystal-field theory in prior studies.

Main Results:

• Successfully distinguished between O-nitrito and N-nitro binding modes based on nitrite nitrogen hyperfine coupling.
• Identified a unique HYSCORE spectral fingerprint for the O-nitrito binding mode.
• Demonstrated the applicability of EPR/HYSCORE for analyzing nitrite coordination in various heme systems.

Conclusions:

• EPR/HYSCORE spectroscopy provides a robust method for determining nitrite ligation modes in heme proteins.
• The identified spectral fingerprint offers direct evidence for O-nitrito binding.
• This tool enhances the understanding of nitrite reactivity in enzymes and catalysts.