Journal
BIOCHEMISTRY
Volume 44, Issue 51, Pages 16785-16795Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi051305z
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Funding
- NCRR NIH HHS [P20 RR15566, P20 RR015566] Funding Source: Medline
- NHLBI NIH HHS [R01-HL065217] Funding Source: Medline
- NICHD NIH HHS [P01-HD0805] Funding Source: Medline
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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 (similar to 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 Cys(52) 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 Cys(52) thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.
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