NO* binds human cystathionine β-synthase quickly and tightly

Area of Science:

• Biochemistry
• Enzymology
• Redox signaling

Background:

• Human cystathionine β-synthase (CBS) contains a heme that regulates its activity.
• Nitric oxide (NO•) and carbon monoxide (CO) can bind to the reduced heme iron, inhibiting CBS.
• Previous studies reported low affinity of NO• for CBS heme, contrasting with its proposed physiological role.

Purpose of the Study:

• To reconcile the discrepancy in NO• binding affinity to human CBS heme.
• To investigate the kinetics and thermodynamics of NO• interaction with recombinant human CBS.
• To elucidate the mechanism of NO•-mediated regulation of CBS activity.

Main Methods:

• Recombinant human CBS was used.
• Static and stopped-flow UV-visible absorption spectroscopy were employed.
• Binding affinity (Kd) and kinetic rate constants (k on, k off) for NO• interaction were determined.

Main Results:

• NO• binds tightly to the ferrous CBS heme with an apparent Kd ≤ 0.23 μm.
• NO• exhibits rapid binding (k on ∼ 8 × 10³ m⁻¹s⁻¹) and slow dissociation (k off ∼ 0.003 s⁻¹) from CBS.
• NO• binding kinetics are >100-fold faster than CO, suggesting a different regulatory mechanism.

Conclusions:

• The heme of human CBS binds NO• quickly and tightly, providing a mechanistic basis for in vivo regulation.
• These findings support a significant role for NO• in regulating CBS activity and cellular signaling.
• The study reinforces the interplay between gasotransmitters (NO•, CO, H2S) in cellular communication.