The Heme of Cystathionine β-synthase Likely Undergoes a Thermally Induced Redox-Mediated Ligand Switch

Abstract

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.

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

“…Our previous results indicated that heme does not function in redox sensing, ligand binding, or catalysis but rather supported the structural role of heme in proper folding and/or subunit assembly (22,28,(32)(33)(34)(35). With respect to this hypothesis and the presented data, heme saturation could serve as a marker for properly folded CBS and could be indicative of the activity of purified CBS.…”

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding

“…As we mentioned before in this studies, we named the new species as the P420 form if its ferrous CO-binding absorbance spectrum showed a Soret maximum around 420 nm. After heating at 48 for more than 30 min, intensity of ℃ the Soret band decreased obviously. But the existence of a 416 nm Soret band in absorbance spectrum and a 419 nm in CD spectrum (as described later, Figure 3C) indicated the heme of P450 2C8 was still buried in the heme pocket at 48 .…”

“…But the existence of a 416 nm Soret band in absorbance spectrum and a 419 nm in CD spectrum (as described later, Figure 3C) indicated the heme of P450 2C8 was still buried in the heme pocket at 48 . When the temperature was cooled ℃ from 48 to 20 ℃ , the intensity of the Soret band i ℃ ncreased apparently without wavelength shift ( Figure 3A inset).…”

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

“…In contrast to CO, NO ⅐ binding to Fe(II)-CBS results in a pentacoordinate iron-nitrosyl Fe(II)NO ⅐ -CBS species with a Soret maximum at 394 nm, indicating the loss of both endogenous axial ligands (34 (32). In addition, Fe(II)-CBS can slowly decay to an inactive Fe(II)424-CBS form with a Soret maximum at 424 nm, in which the cysteine is replaced by an unidentified neutral ligand (35,36). Although Fe(II)424-CBS formation is promoted under nonphysiological conditions such as high temperature (36), the biological significance of this process is not clear.…”

“…In addition, Fe(II)-CBS can slowly decay to an inactive Fe(II)424-CBS form with a Soret maximum at 424 nm, in which the cysteine is replaced by an unidentified neutral ligand (35,36). Although Fe(II)424-CBS formation is promoted under nonphysiological conditions such as high temperature (36), the biological significance of this process is not clear. We have demonstrated recently that Fe(II)-CBS has the ability to reduce nitrite (NO 2 Ϫ ) to NO ⅐ , leading to Fe(II)NO ⅐ -CBS formation, suggesting a possible new role for CBS in NO ⅐ signaling (37).…”