Biochemistry, Department of


Document Type


Date of this Version



J Inorg Biochem. 2009 May ; 103(5): 689–697.


Copyright 2009 Elsevier. Used by permission.


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.