| Human cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique among PLP-dependent enzymes in that heme is required for maximal activity, although the function of heme in this enzyme has yet to be elucidated. The goals of this work are: (1) to provide insight into the role of the heme cofactor in CBS by probing specific structure/function relationships that relate the heme and the enzyme's active site; and (2) to more broadly explore low-spin heme-thiolate centers using computational methods, and relating their spectroscopic and functional behavior to those of other heme-thiolate centers.; The work described herein revealed that the CBS heme is only stable to reduction under very specific conditions. The CBS heme is a low-spin, cysteine(thiolate)-coordinated center, a rarity among heme proteins. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, show that at 4 °C and pH 9, Fe(II) CBS is the only low-spin heme-thiolate in which the heme retains the cysteinate ligand in the reduced state. At more moderate temperatures, including those in the physiological range, the CBS heme undergoes a redox-mediated ligand switch. Spectroscopic interrogation suggests that upon reduction at pH 9 and 37 °C, the native cysteinate ligand of the CBS heme is displaced by a neutral donor. Functional assays have shown that this form of CBS, termed "Fe(II) CBS-424," is inactive. At low pH, Fe(II) CBS reoxidizes by an apparent proton-gated electron-transfer mechanism that likely lowers the redox potential of the CBS heme. The physiological relevance of these processes is not known; however, inactivation of CBS via Fe(II) CBS-424 formation serves as evidence for communication between the heme and the PLP active site and thus a regulatory role for the heme in this enzyme. In order to theoretically probe the behavior of the CBS heme and other low-spin heme-thiolates, DFT calculations were used to demonstrate that significant sulfur character in the ground state of these heme centers as well as hydrogen-bonding interactions modulate the electronic structure of the heme-thiolate moiety. |
