Heme-based CO sensors CooA Cystathionine b-synthase (CBS) 1
Carbon monoxide (CO): Simple! Heme Fe(II) complex, Metal complex NO, H 2 S : Complicated! Heme iron complex, Protein modification, Other molecules 2
CO interacts with heme Fe(II), and other metal complexes (never interacts with heme Fe(III) and other non-metal molecules NO interacts with heme Fe(II), heme Fe(III), amino acids, proteins and other molecules H2S interacts with heme Fe(III), amino acids, proteins and other molecules 3
Nature Rev. Drug Discov. 9, 728 (2010) The therapeutic potential of carbon monoxide Carbon monoxide (CO) is increasingly being accepted as a cytoprotective and homeostatic molecule with important signalling capabilities in physiological and pathophysiological situations. The endogenous production of CO occurs through the activity of constitutive (haem oxygenase 2) and inducible (haem oxygenase 1) haem oxygenases, enzymes that are responsible for the catabolism of haem. Through the generation of its products, which in addition to CO includes the bile pigments biliverdin, bilirubin and ferrous iron, the haem oxygenase 1 system also has an obligatory role in the regulation of the stress response and in cell adaptation to injury. This Review provides an overview of the physiology of CO, summarizes the effects of CO gas and CO-releasing molecules in preclinical animal models of cardiovascular disease, inflammatory disorders and organ transplantation, and discusses the development and therapeutic options for the exploitation of this simple gaseous molecule. 4
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FIGURE 2 Interactions between the three gas-generating systems. Carbon monoxide (CO), nitric oxide (NO) and hydrogen sulphide (H 2 S) are generated by endogenous enzymatic systems with complex interrelationships. Pharmaceutical development has taken advantage of these systems to design exogenous molecules to simulate those generated endogenously. The mitochondrion is the common organelle targeted by all three gases, which also modulate oxygen (O 2 ) reactivity and consumption. CBS, cystathionine β-synthase; CO-RM, CO-releasing molecule; CSE, 6 cystathionine γ-lyase; HO1, haem oxygenase 1; NOS, NO synthase; ROS, reactive oxygen species.
FIGURE 4 Effects of inhaled carbon monoxide on Plasmodium bergheiinduced blood brain barrier disruption and parenchymal brain haemorrhage. Blood brain barrier disruption in C57Bl/6 mice infected with P. berghei was assessed by Evans blue dye extravasation indicated by the blue shading in infected mice exposed to air or carbon monoxide (CO) plus air. CO was administered at 250 ppm on days 3 and 4 after infection, and brains were harvested 6 12 days post-infection at the time of onset of cerebral malaria in the air-treated controls. Images are reproduced, with permission, from Nature Medicine Ref. 81 (2007) Macmillan Publishers Ltd. All rights 7
FIGURE 5 CO-RM2 rescues HO1-deficient mice from arterial thrombosis after aortic transplantation. A Administration of carbon monoxide-releasing molecule 2 (CO-RM2) before and after transplantation resulted in ~60% of mice surviving, compared with 0% survival in mice treated with inactive CO-RM (ico-rm). B Immunopathology shows no arterial thrombus in allogeneic grafts from Hmox1 +/+ mice treated with either ico-rm2 (a) or CO-RM2 (b). This lack of thrombus formation, in part owing to haem oxygenase 1 (HO1)-derived CO, was lost in ico-rm2-treated Hmox1 / mice (c), in contrast to Hmox1 / mice treated with CO-RM2 in which lack of thrombus formation was restored (d). Images are reproduced, with permission, from Ref. 102 (2009) American Society for Investigative Pathology. 8
Review: Signaling by Gasotransmitters Sci. Signal, 28 April 2009, Vol. Issue 68, re2 9
Nature Medicine 17, 1391 (2011) 10
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Trends. Biochem. Sci. 31, 614 (2006) 12
Ribbon diagram of the structure of CooA in its inactive Fe[II] state and of CAP in its active, camp-bound state. Candace M. Coyle et al. J. Biol. Chem. 2003;278:35384-35393 2003 by American Society for Biochemistry and Molecular Biology
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[A] [B] camp camp camp Pro 2 Pro 2 Fe(III) Fe(II) Fe(II) O C Cys 75 His 77 His 77 camp activates expression of catabolite-sensitive operons [C] Fe Fe Fe Fe CO Ala 2 Ala 2 NH NH Fe(III) Fe(II) Fe(II) O C His 82 His 82 His 82 CO activates expression of coo genes Fig. 11 15
J. Am. Chem. Soc. 133, 18816 (2011) 16
A new gaseous signaling molecule emerges: Cardioprotective role of hydrogen sulfide Copyright 2007 by the National Academy of Sciences Fig. 1. Summary of the physiological actions of hydrogen sulfide (H2S) Lefer, David J. (2007) Proc. Natl. Acad. Sci. USA 104, 17907-17908 17
Box1. H 2 S biosynthesis 18
H2S is produced from cysteine or homocysteine by the P5P-dependant transsulfuration enzymes, CBS (cystathionine beta-synthase) and CGL (cystathionine gamma-lyase, aka CSE), as well as by the enzymes 3MST (3-mercaptopyruvate sulfurtransferase), CAT (cysteine aminotransferase) and possibly others (e.g. 3MP) [3]. Murine studies have shown that in the liver, CBS is only responsible for 3% of H2S production by the transsulfuration pathway, whereas in the kidney and brain the majority of H2S comes from the CBS reaction [4], the extent of which is dependent upon its allosteric activation by SAMe [5]. Notably CBS dominance in the brain is also suggested by higher relative levels of cystathionine in the rodent and primate brain [6]. Production of H2S is likely to be altered under hyperhomocysteinemic conditions where the relative contribution of CBS to H2S formation may decrease and CGL increase [5]. H2S is released either directly after enzymatic production or from sulfur stores in response to acidic conditions or reducing agents [3]. H2S is metabolised or detoxified mainly through oxidation where it is converted first to sulphite by the enzyme sulfite reductase, then to sulphate by the enzyme sulphite oxidase (molybdenum cofactor), and finally excreted in urine. 19
Fig. 2. Modular organization of human cystathionine β-synthase and structure of the truncated enzyme. (A) Schematic depiction of the modular organization of human cystathionine β-synthase. The boundaries for the various domains are indicated as are the two CBS domains in the regulatory C-terminal region. (B) The structure of dimeric human cystathionine β-synthase lacking the C-terminal regulatory domain. The color code employed in (A) is retained to depict the heme and PLP (pyridoxal phosphate) binding domains. The heme is shown in red and the PLP in yellow. The figure was generated using the pdb file 1M54. 20
Fig. 3. Close up of the heme-binding domain and its conservation in other cystathionine β-synthases. (A) The axial ligands of the heme, C52 and H65, are shown along with the conserved residues in the immediate vicinity of H65. (B) Conservation of sequences in the immediate vicinity of the heme ligand residues in cystathionine β-synthase, The numbers at the top refer to those for cysteine and histidine in the human sequence. The presence of heme is established in the rat and human proteins but has not been tested in Anopheles gambiae, Drosophila melanogaster or in Dictyostelium discoides. 21
[A] [C] N N H65 Fe N N C52 PLP K119 CXXC 416 468 486 543 CBSI N C 1 70 411 551 heme domain catalytic core regulatory domain CBSII CO His 65 His 65 His 65 Fe(II) C O Cys 52 [B] Fe(III) Fe(II) His 66 Cys 52 Cys 52 His 67 Pro 64 His 65 NO His 65 Fe(II) O N Fe(II) His 65 Cys 52 Cys 52 N O Cys 52 pyridoxal-phosphate (PLP); S-adenosyl-L-methionine (adomet). 22
Cystathionine-β-synthase, also known as CBS, is an enzyme (EC 4.2.1.22) that in humans is encoded by the CBS gene. It catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine: L-serine + L-homocysteine = L-cystathionine + H 2 O CBS uses the cofactor pyridoxal-phosphate (PLP) and can be allosterically regulated by effectors such as the ubiquitous cofactor S-adenosyl-L-methionine (adomet). This enzyme belongs to the family of lyases, to be specific, the hydro-lyases, which cleave carbon-oxygen bonds. In mammals, CBS is a highly regulated enzyme, which contains a heme cofactor that functions as a redox sensor, that that can modulate its activity in response to changes in the redox potential. If the resting form of CBS in the cell has Fe(II) heme, the potential exists for activating the enzyme under oxidizing conditions by conversion to the Fe(III) state. The Fe (II) form of the enzyme is inhibited upon binding CO or NO, whereas enzyme activity is doubled when the Fe (II) is oxidized to Fe (III). The redox state of the heme is ph dependent, with oxidation of Fe (II)-CBS to Fe (III)-CBS being favored at low ph conditions. Since mammalian CBS contains a heme cofactor, whereas yeast and protozoan enzyme from Trypanosoma cruzi do not have heme cofactors, researchers have speculated that heme is not required for CBS activity. Wikipedia 24
Oscillation Mechanism Forebrain NPAS2 E-box BMAL1 per, cry Mouse brain BMAL1 NPAS2 HO heme heme heme CO E-box E-box E. M. Dioum et al., Science 298, 2385 (2002) 25
Reciprocal Regulation of Circadian Clock and Heme Biosynthesis PER2 PER2 Per2 NPAS2 BMAL1 Fe HO PER2 Fe CO BMAL1 NPAS2 Fe Fe PER2 NPAS2 BMAL1 Alas1 Kaasik et al. Nature 2004 ALAS1 26
TABLE 2 Pharmacological classification of CO gas and CO- RMs 27
Carbon Monoxide-releasing Antibacterial Molecules Target Respiration and Global Transcriptional Regulators J. Biol. Chem. 284, 4516 (2009) Ru(CO) 3 Cl(glycinate) 28