Cobalt cystathionine β-synthase: a cobalt-substituted heme protein with a unique thiolate ligation motif.

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.