Heme oxygenase

3 3

Cloning, expression and purification

E. coli K-12 genome

cloning

pET21c

overexpression Full-length YddV

97

66

45

(kDa)

54 kDa purification

YddV contains a b-type heme with 1 : 1 stoichiometry.

33

Iron Protoporphyrin IX, heme b Prosthetic group A flat and planar structure. Itself Toxic O2

-., H2O2 are formed.

Heme a Heme a is a form of heme found In cytochromes a and a3.

Heme is regulated by the rate of synthesis and rate of degradation via the rate-limiting enzymes ALA synthase and HO-1. When heme is released by the mitochondria to the cytoplasm and exceeds the physiological need, it will cause suppression of ALA synthesis and/or inhibit ALA transport to the mitochondria; however, if heme is further increased it will cause induction of HO-1 and enhance the rate of its degradation.

HO-1: Induced

HO-2: Constitutive

Pharm. Rev. 60, 79 (2008)

Heme oxygenase Fe(II) forms of verdoheme in a complex with human heme oxygenase-1.

J. Physiol. 589, 3055 (2011) Figure 1. Cartoon illustrating some of the proposed mechanisms by which CO exerts its effect on identified ion channels Each of the molecularly defined ion channels known to be regulated by CO are schematically represented at the top of the figure. These are: the large conductance calcium- and voltage-activated K+ channel (BKCa); the epithelial Na+ channel (ENaC); the tandem P domain K+ channel (TREK-1); the ATP-gated P2X receptors (P2X2 and P2X4); the voltage-activated K+ channel (Kv2.1); and L-type Ca2+ channels. Also shown is the O2-dependent generation of CO from haem by haemoxygenase enzymes (HO), CO-dependent generation of cGMP by soluble guanylyl cyclase (sGC) activation and CO-dependent generation of mitochondrial reactive oxygen species (ROS).

Mammalian Mechanisms of Iron Homeostasis

Biochemistry 51, 5705 (2012)

Fig. 1. Iron absorption, distribution, and recycling in

the body and quantitative exchange of iron between

body iron sources. Body iron levels are maintained by

daily absorption of ~1-2 mg of dietar iron to account

for obligatory losses of a similar amount of iron

through sloughing of mucosal and skin cells,

hemorrhage, and other losses. Approximately 4 mg of

iron is found in circulation bound to Tf, which

accounts for 0.1% of the total body iron. Majority of

the body iron is found in the erythroid compartment of

bone marrow and in mature erythrocytes contained

within the heme moiety of the

hemoglobin. Splenic reticuloendothelial macrophages,

which recycle iron from senescent red blood cells,

provide iron for the new red blood cell synthesis. Tf

delivers iron to developing erythroid precursors, as

well as to other sites of iron utilization. Liver

hepatocytes store iron in ferritin shells. During

pregnancy, 250 mg of iron is transported across the

placenta to the fetus. The distribution of iron in the

body is altered in iron deficiency and iron overload

(see text).

Biochem. Pharm. 80, 1895 (2010)

Bach 1: Heme sensor protein

6 CP motifs: Different heme affinity?

(heme regulatory motif: HRM)

Potential heme binding sites

EMBO J. 20, 2835 (2001)

Transcription regulator protein BACH1 is a protein that in humans is encoded by the BACH1 gene. This gene encodes a transcription factor that belongs to the cap'n'collar type of basic region leucine zipper factor family (CNC-bZip). The encoded protein contains broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domains, which is atypical of CNC-bZip family members. These BTB/POZ domains facilitate protein-protein interactions and formation of homo- and/or hetero-oligomers. When this encoded protein forms a heterodimer with MafK, it functions as a repressor of Maf recognition element (MARE) and transcription is repressed. Wikipedia

Regulation of iron recycling by a Bach1/Nrf2 dependent transcriptional control mechanism. Erythrophagocytosis or uptake of extracellular hemoglobin via the hemoglobin scavenging receptor CD163 cause increased intracellular heme levels (1). Heme accumulation leads to the release of the transcriptional repressor Bach1 from the ARE/MARE enhancer, KEAP1 degradation and subsequent nuclear accumulation of the transcriptional activator Nrf2. Nrf2 binding to small MAFs (SM) enhances the transcription of heme-oxygenase 1 (HO1), the ferritins and FPN1 (2). HO1 releases inorganic iron from the heme moiety (3) which inhibits IRP1 and IRP2 binding to the FPN1 and ferritin IREs to promote mRNA translation. Free iron is

Haematologica

95, 1261 (2010)

Nature Medicine 17, 1391 (2011)

Pharm. Rev. 60, 79 (2008)

J. Physiol. 589, 3055 (2011) Figure 1. Cartoon illustrating some of the proposed mechanisms by which CO exerts its effect on identified ion channels Each of the molecularly defined ion channels known to be regulated by CO are schematically represented at the top of the figure. These are: the large conductance calcium- and voltage-activated K+ channel (BKCa); the epithelial Na+ channel (ENaC); the tandem P domain K+ channel (TREK-1); the ATP-gated P2X receptors (P2X2 and P2X4); the voltage-activated K+ channel (Kv2.1); and L-type Ca2+ channels. Also shown is the O2-dependent generation of CO from haem by haemoxygenase enzymes (HO), CO-dependent generation of cGMP by soluble guanylyl cyclase (sGC) activation and CO-dependent generation of mitochondrial reactive oxygen species (ROS).

Carbon monoxide (CO): Heme iron complex, Metal complex NO, H2S : Heme iron complex, Protein modification, Other molecules

Nat. Rev. Drug Discov. 9, 728 (2010)

Inorg. Chem. 49, 3602 (2010)

NADPH P450-reductase

NADPH P450-reductase

Cytochrome P450: Cysteine-S binding to Fe(II) heme is important for

activation of O2.

Cytochrome c, Cytochrome b5: Electron-transfer relating heme proteins.

Myoglobin and hemoglobin: Histidine-midazole binding to Fe(II) heme

is important for O2 storage and O2 carrier, respectively. 23

24

R: substrate

R: substrate

R: substrate

R: substrate

R: substrate

Shunt reaction

25

26

Catalysis of Nitric Oxide Synthase (NOS)

O O

P450: Fe(III)heme-Cys, Reductase NOS: Fe(III)heme-Cys, Calmodulin H4B, fused enzyme with reductase, dimer 27

Inorg. Chem. 49, 3602 (2010)

Narrow space on the heme distal side for a substrate to bind.

The O2 molecule would be bent toward the water molecule.

J. Inorg. Biochem. 113, 102 (2012)

Schematic representation of the role of CO in biological systems. Perturbations in the levels of CO influence a broad spectrum of metabolic pathways. Although CO, like NO, acts to stimulate cGMP, it differs from NO in many functions. For example, CO produces vasorelaxation by stimulating K channels (at the α subunit, but NO acts at the β subunit), increases angiogenesis, and is antiapoptotic and anti-inflammatory. CO may stimulate NO release from the NO binding sites of the heme molecules.

Role of heme oxygenase and carbon monoxide in lung diseases. Heme oxygenase (HO) generates biliverdin IXα, ferrous iron, and carbon monoxide (CO) from the oxidation of heme. Exhaled CO reflects active heme metabolism. Inflammation, oxidative stress, and apoptosis represent an axis of disease, against which both endogenous HO activity and exogenous CO exert protective effects. CO may inhibit both inflammation and apoptosis. The toxicological properties of CO imply increased pro-oxidant activity; however, the pro-oxidant/and antioxidant consequences of CO in the physiological range remain unclear. The bile pigments biliverdin IXα and bilirubin IXα have demonstrated antioxidant properties, though their prospective roles in modulation of inflammation and apoptosis are currently under investigation. Iron (Fe) released from HO activity returns to a transient chelatable pool, where it may potentially promote oxidative stress and apoptosis. Induction of ferritin synthesis and sequestration of the released iron into ferritin may represent one possible detoxification pathway that limits the potential of iron in pro-apoptotic and pro-oxidative processes. Slebos et al. Respiratory Research 2003 4:7

CO in smoking?

Schematic representation of the role of bilirubin in biological systems, representing the antioxidant properties of the bile pigment. Bilirubin is produced from the reduction of biliverdin by the cytosolic enzyme biliverdin reductase. Bilirubin, like all compounds, is toxic at high concentrations, e.g., kernicterus in newborns. However, bilirubin has been shown to prevent adhesion molecule expression and neutrophil adhesion and inhibits ROS and NADPH oxidase activity.

Schematic representation of HO-1-derived CO and bilirubin in the regulation of oxidative stress and cell survival. Up-regulation of HO-1 by pharmacological agents or by gene transfer or as a result of stress leads to an increase in heme degradation and the generation of CO and bilirubin. This process increases heme turnover with a resultant effect of decreasing inducible enzymes such as iNOS, but not eNOS. Simultaneously, CO or bilirubin or both enhance antiapoptotic, antioxidant, and signaling molecules. HO-1-derived CO or bilirubin may directly increase EC-SOD or, via their activation, transcriptional factors. EC-SOD scavenges , decreases formation of ONOO-, and limits oxidative and nitrative stress.

Cardiovascular drugs that increase HO-1 expression and HO activity. These represent a broad spectrum of chemical structures, illustrating the disparity of compounds that induce HO-1 (there is, however, a commonality among the structures, such as the phenol ring, which may be considered as a major activator of the HO-1 promoter region) and increase HO-1 mRNA, proteins, and total HO activity. Other drugs are not included in the scheme, such as CoPP with a 4-carbon and nitrogen ring. In addition, sildenafil citrate (Viagra) and tadalafil (Cialis) either increase total HO-1 activity and CO levels.

Acute kidney injury (AKI) is enhanced by several factors, including drugs and ischemia, all of which result in increased levels of IL-1, IL-6, TNFα, and angiotensin II. These factors activate a number of signaling factors, including AP-1, AP-2, NF-κB, Ets, and Nrf2, leading to a rapid increase in HO-1 mRNA levels. Drugs that increase HO-1, such as CoPP, gene transfer or gene targeting to specific renal structures, have been shown to attenuate acute kidney injury.

Schematic representation of the role of HO-1 in hypertension. Induction of HO-1 leads to perturbations in renal arachidonic acid metabolism, including inhibition of the heme-containing thromboxane A2 (TxA2) synthase and CYP4A enzymes that are responsible for the formation of TxA2 and 20-HETE, respectively; both are potent renal vasoconstrictors whose bioactivity leads to increased renal vascular resistance, vascular tone, and blood pressure. Alternatively, HO-1 induction leads to increased production of CO, which, in addition to its ability to inhibit heme-containing enzymes, also acts as a vasodilator and regulator of ion channels, thus contributing to antihypertensive mechanisms, including increased vasodilation and natriuresis.

Schematic representation of the role of HO-1 regulation in the brain. Heme metabolism in the brain is under separate control from heme metabolism in other tissues. The brain is unique, and oxidative stress and hypoxia have been shown to be involved in neurodegenerative processes. HO-1 induction will contribute to the inhibition of oxidative stress, a known factor in numerous neurodegenerative diseases, including Alzheimer's disease.

Mitochondrial HO-1. Schematic representation of the effects of HO-1 induction on the caspase pathway leading to apoptosis, in which HO-1 induction decreases caspase 3 and 9. In addition, increased HO-1 levels result in increased CO production and activation of p38 MAPK and the STAT-3 pathway. Similarly, increased HO-1 expression increases pBAD levels, resulting in increased cell survival and mitochondrial function (Olszanecki et al., 2007; Turkseven et al., 2007

Figure 1. Gram-negative bacteria heme-uptake systems. (a) The HemR receptor of Y. enterocolitica. Heme/Hb binds the HemR receptor and heme is transported through the receptor channel in a TonB-dependent manner. See Figure 2 for more details. Other HemR-type receptors include ShuA from Shigella, ChuA from E. coli O157:H7, and HmuA from Y. pestis [15], [16] and [18]. (b) The HmbR receptor of H. influenzae. The receptor is highly adapted to one or two heme–protein complexes. (c) The neisserial HpuAB receptor consists of two outer membrane proteins HpuA, a membrane lipoprotein, and HpuB, a Ton-dependent receptor. (d) Hemophores are secreted extracellularly where they bind heme. The hemophere delivers the bound heme to a specific outer membrane receptor (HasR). The heme is transported into the cell by a TonB-dependent manner. The HasA family of hemophores are found in Y. pestis, P. aeruginosa, P. fluorescens and S. marcescens[35••]. The hemophore HxuA, which binds hemopexin, is found in H. influenzae[38]. The ABC protein is a membrane-associated ATPase belonging to the ATP-binding cassette protein superfamily [31]. The ABC protein is involved in heme transport from the periplasm to the cytoplasm. IM, inner membrane; OM, outer membrane; N, amino terminus; COOH, carboxyl terminus.

Current Opinion Microbiol. 3, 215 (2000) Heme iron complex is toxic. Bacteria need Fe as nutrition.

Nat. Prod. Rep. 2007, 24, 511 Angela Wilks

A proposed protective role for HO-1 in the regulation of NO and iNOS in atherosclerosis. HO-1 inhibits ROS and iNOS and subsequently inhibits various steps in the pathogenesis of atherosclerosis, including endothelial cell activation, migration of leukocytes, oxidation of LDL, angiotensin- and inflammatory cytokine-mediated increases in smooth muscle cell proliferation, foam cell formation, and plaque formation.

Oxygen Sensing: It's a Gas!

Science 306, 2050 (2004) Perspective

48

Oxygen sensations. In response to a decrease in oxygen tension (hypoxia), BK channel activity is

blocked and the channel closes, leading to depolarization of glomus cells in the carotid body. Oxygen

sensing depends on the cooperation of BK channels with hemoxygenase-2 (HO-2) and carbon

monoxide (CO). In the presence of oxygen, HO-2 uses electrons from NADPH cytochrome-P450

reductase and intracellular heme to generate CO, which in turn exerts a tonic excitatory influence on

neighboring BK channels, which remain open. Hypoxia disrupts the production of CO and

consequently inhibits BK channel activity. Only two of the multiple Ca2+ binding sites of BK

channels are shown. It is not known how many CO molecules are required to stimulate BK channel

activity. HO-2 may interact directly with the BK channel.

Hemeoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel Science 306, 2093 (2004) Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2–dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.