Dynamics of Carbon Monoxide Binding to Cystathionine β-Synthase

Mrinalini Puranik,Colin L Weeks,Dorothee Lahaye,Ömer Kabil,Shinichi Taoka,Steen Brøndsted Nielsen,John T Groves,Ruma Banerjee,Thomas G Spiro

doi:10.1074/jbc.m600246200

Abstract

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.

Highlights

Enzyme is inhibited upon binding carbon monoxide (CO) or nitric oxide [5, 6], whereas activity is doubled when the Fe(II) is oxidized to Fe(III) [7]
We have investigated CO binding to truncated CBS with the aim of elucidating the mechanism of regulation by this gas
Our principal findings are that the dimer displays equilibrium anticooperativity in CO binding, that the rate of CO binding is controlled by pre-dissociation of the endogenous Cys-52 ligand, and that rebinding of Cys-52 is relatively slow and independent of pH, between 7.6 and 10.5, implying a perturbed pKa for the dissociated Cys-52

Summary

MATERIALS AND METHODS

Enzyme Purification and CO Equilibrium Constant Measurements— The truncated dimer, CBS-⌬C143, was purified as described previously [11]. The binding constants for CO for the truncated enzyme were determined exactly as described for the full-length enzyme [5]. Sample Preparation—For Raman spectroscopy, aliquots of Fe(III)CBS protein were transferred to NMR tubes and diluted using specified buffers to achieve a typical concentration of 25 ␮M in heme. To prepare the CO-bound form of CBS following the reduction step above, the samples were immediately purged with CO for 3–5 min. For the rapid mixing experiments, Fe(II)-CBS for the stopped flow experiments was prepared with the same procedure as for Raman experiments. The final concentration was 14 ␮M before mixing. CO-saturated buffers for use in rapid mixing experiments were prepared by first degassing with alternate application

Dynamics of CO Binding to CBS

RESULTS

DISCUSSION