title: Cobalt cystathionine β-synthase: a cobalt-substituted heme protein with a unique thiolate ligation motif.
abstract: Human cystathionine β-synthase (hCBS), a key enzyme in the trans-sulfuration pathway, catalyzes the condensation of serine with homocysteine to produce cystathionine. CBS from higher organisms is the only known protein that binds pyridoxal-5'-phosphate (PLP) and heme. Intriguingly, the function of the heme in hCBS has yet to be elucidated. Herein, we describe the characterization of a cobalt-substituted variant of hCBS (Co hCBS) in which CoPPIX replaces FePPIX (heme). Co(III) hCBS is a unique Co-substituted heme protein: the Co(III) ion is 6-coordinate, low-spin, diamagnetic, and bears a cysteine(thiolate) as one of its axial ligands. The peak positions and intensities of the electronic absorption and MCD spectra of Co(III) hCBS are distinct from those of previously Co-substituted heme proteins; TD-DFT calculations reveal that the unique features arise from the 6-coordinate Co bound axially by cysteine(thiolate) and a neutral donor, presumably histidine. Reactivity of Co(III) hCBS with HgCl(2) is consistent with a loss of the cysteine(thiolate) ligand. Co(III) hCBS is slowly reduced to Co(II) hCBS, which contains a 5-coordinate, low-spin, S = 1/2 Co-porphyrin that does not retain the cysteine(thiolate) ligand; this form of Co(II) hCBS binds NO((g)) but not CO((g)). Co(II) hCBS is reoxidized in the air to form a new Co(III) form, which does not contain a cysteine(thiolate) ligand. Canonical and alternative CBS assays suggest that maintaining the native heme ligation motif of wild-type Fe hCBS (Cys/His) is essential in maintaining maximal activity in Co hCBS. Correlation between the coordination structures and enzyme activity in both native Fe and Co-substituted proteins implicates a structural role for the heme in CBS.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, Wisconsin 53706, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Smith
authors.forename: Tomas
authors.initials: T
authors.lastname: Majtan
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Freeman
authors.forename: Yang
authors.initials: Y
authors.lastname: Su
authors.forename: Jan P
authors.initials: JP
authors.lastname: Kraus
authors.forename: Judith N
authors.initials: JN
authors.lastname: Burstyn
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism*; D056831:Coordination Complexes / Q000737:chemistry / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic
chemical_list: D056831:Coordination Complexes; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D003035:Cobalt; D006418:Heme; D006639:Histidine; D011732:Pyridoxal Phosphate; D003541:Cystathionine beta-Synthase; D003545:Cysteine