ABSTRACT
Cystathionine ß-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly Drosophila melanogaster (DmCBS) and the CO/NO gas binding reactions of DmCBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), DmCBS is intrinsically highly active and is not regulated by AdoMet. The DmCBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The DmCBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe(II) heme in DmCBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and DmCBS Fe(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme.
Subject(s)
Cystathionine beta-Synthase/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster/enzymology , Heme/chemistry , Amino Acid Sequence , Animals , Carbon Monoxide/chemistry , Conserved Sequence , Cystathionine beta-Synthase/genetics , Drosophila Proteins/genetics , Enzyme Stability , Evolution, Molecular , Humans , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Nitric Oxide/chemistry , Oxidation-Reduction , Protein BindingABSTRACT
CooA is a heme-dependent CO-sensing transcription factor that has three observable heme coordination states. There is some evidence that each CooA heme state has a distinct protein conformation; the goal of this study was to characterize these conformations by measuring their structural stabilities through guanidine hydrochloride (GuHCl) denaturation. By studying the denaturation processes of the Fe(III) state of WT CooA and several variants, we were able to characterize independent unfolding processes for each domain of CooA. This information was used to compare the unfolding profiles of various CooA heme activation states [Fe(III), Fe(II), and Fe(II)-CO] to show that the heme coordination state changes the stability of the effector binding domain. A mechanism consistent with the data predicts that all CooA coordination states and variants undergo unfolding of the DNA-binding domain between 2 and 3 M GuHCl with a free energy of unfolding of approximately 17 kJ/mol, while unfolding of the heme domain is variable and dependent on the heme coordination state. The findings support a model in which changes in heme ligation alter the structural stability of the heme domain and dimer interface but do not alter the stability of the DNA-binding domain. These studies provide evidence that the domains of transcription factors are modular and that allosteric signaling occurs through changes in the relative positions of the protein domains without affecting the structure of the DNA-binding region.
