Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands: The role of heme ligand switching in redox regulation

Abstract

In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424 nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449 nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is −350 ± 4 mV and is substantially lower than the value of −287 ± 2 mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.

Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones

“…These promising results from E. coli crude extracts encouraged us to attempt to purify the mutants to homogeneity. Several previous attempts to purify CBS mutants resulted in low yields or failed completely due to the unstable mutant conformations, their extreme tendency to aggregation, and heme loss (12,30,31). The presence of chemical chaperones during CBS expression helped to overcome these problems, resulting in homogeneous mutant CBS enzymes as active as WT.…”

Relative Contributions of Cystathionine β-Synthase and γ-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

“…Expression and Purification of hCBS, yCBS, and hCSE-Recombinant full-length hCBS and yeast CBS (yCBS) were purified as described previously (22,23). The expression plasmids encoding hCBS and yCBS were provided by Drs.…”

NO• Binds Human Cystathionine β-Synthase Quickly and Tightly

“…The heme iron of human CBS both in the oxidized and reduced state is low spin, hexa-coordinate, being axially bound by the endog-enous ligands His-65 and Cys-52. This heme displays a remarkably low reduction potential (Ϫ350 mV versus standard hydrogen electrode) (13) and acts as a redox-dependent gas sensor because of its ability to react with O 2 and to bind CO and NO ⅐ in the reduced form. Despite the low reduction potential and the fact that most studies on the CBS heme reactivity were carried out in the presence of a large excess of the potent chemical reductant sodium dithionite, it has been recently shown that the diflavin enzyme methionine synthase reductase is able to reduce the CBS heme with NADPH as the electron donor (14,15), conferring greater physiologic relevance to ferrous CBS and its adducts with CO and NO ⅐ .…”

“…An electrostatic interaction between the Cys-52 thiolate and the guanidinium group of Arg-266 has been identified as a key element in communicating the heme CO binding to the pyridoxal 5Ј-phosphate active site (13,17,18). The reaction with CO proceeds at very low rates showing hyperbolic dependence on CO concentration (14 -16, 19).…”