1

Heme sensors

2

General concepts and examples

Sensor domain Function domain

Protein

structural

changes

Sensor domain

(heme-bound)

Function domain Catalysis,

DNA binding

Heme-responsive heme-sensor proteins

N N

N N

O

O

O

O

Heme as the

1st signal

Fe

3

4

1 138 619

kinase domain (KD) HRI NTD

Cys

His

Cys

His

heme

Inactive Active

heme

eIF2

ATP ADP

eIF2 eIF2 P

Association/dissociation of heme iron regulates the kinase

reaction associated with protein synthesis.

FeII/III heme reduces its own availability in a cell-type-specific way. In erythrocytes, FeII/III heme induces primarily ALAS2 expression, whereas ALAS1 biosynthesis in most tissues (e.g. liver) is inhibited. Inhibition of IRP2 (Homo sapiens) and Irr (B. japonicum) is achieved by FeII/III heme-induced proteasomal degradation of these proteins. This highlights the conservation of the regulatory function of FeII/III heme in different organisms. Through binding of FeII/III heme to heme oxygenase 2 (HO-2), the available amount of FeII/III heme in the cell is reduced. (5-ALA: 5-aminolevulinic acid). 5

Versatile influences of FeII/III heme on transcriptional (transcription repression and activation, miRNA-processing) are indicated for prokaryotes (e.g. Irr from B. japonicum, PpsR from Rhodobacter sphaeroides) and eukaryotes (e.g. HAP1 from Saccharomyces cerevisiae, Bach1 from Homo sapiens). Rhythmically oscillating FeII/III heme levels are reflected in regulatory processes by FeII/III heme during circadian rhythm. Therefore, binding of FeII/III heme in a first step allows sensing concentration changes of CO and NO. (Cry: cryptochrome; DGCR8: DiGeorge syndrome critical region 8).

6

Translation and proteasomal protein degradation are regulated and cross-linked by FeII/III heme at different levels. Inhibition of HRI in erythrocytes increases synthesis of α- and β-globins; these integrate FeII heme during the formation of hemoglobin. Arginyl-tRNA synthetase cross-links processes of translation and proteasomal protein degradation. Through attachment of FeII/III heme to proteins Per2, Bach1, Irr, and IRP2, their own proteasomal degradation is induced. In contrast, degradation processes that are mediated by ATE1, UBR1, UBR2, and arginyl-tRNA synthetase are inhibited by FeII/III heme. (HRI-P: phosphorylated HRI; eIF2(αP): phosphorylated eIF2α).

7

Influences of FeII/III heme on signal transduction pathways, immune responses, and transmembrane ion transport. FeII/III heme binds directly to ion channels, which, in the case of hSlo1, leads to increased vasoconstriction in the brain.49a, b In most reports, changes in signal transduction levels and immune responses are general reactions upon application of FeII/III heme to whole cells or from growth of cells on FeII/III heme-deficient media. Neudesin and neuferricin are the first extracellular, temporary FeIII heme-binding proteins, and show different effects upon FeIII heme application.3b Reports on the cross-link of PKC and MAPK-pathway raise the suggestion of further crossovers not mentioned here. (ERK1/2-P: phosphorylated extracellular signal-regulated kinase 1/2; Raf-P: phosphorylated Raf; MEK1/2: phosphorylated MAP/ERK kinase 1/2; NGF: nerve growth factor).

8

Porphyrin Handbook Vo. 15, Chapter 73 (2011)

9

Specific features of the heme-responsive heme sensor proteins

1. Thiolate as the heme-binding (heme-sensing) site.

2. CP (Cys-Pro) motif or HRM (heme-regulatory motif).

3. Fast heme dissociation rate.

4. Redox-dependent heme binding.

5. Protein flexibility and plasticity.

Handbook of Porphyrin Science, Vol. 15, World Scientific,

pp. 399-460 (2011).

10

11

1 138 619

kinase domain (KD) HRI NTD

Cys

His

Cys

His

heme

Inactive Active

heme

eIF2

ATP ADP

eIF2 eIF2 P

Association/dissociation of heme iron regulates the kinase

reaction associated with protein synthesis.

12

Cysteine Thiolate is Heme Fe(III) Sensing/Binding

Site of Heme Sensor Proteins

HRI Cys409/His119(His120) (CP motif)

DHR51 Cys/His

DGCR8 Cys352 (CP motif)

RjIrr Cys29 (CP motif)

hcArgRS Cys115

E75 Cys396/His574

Rev-erb Cys418/His602

Rev-erbb Cys384/His568

NPAS2 Cys170/His119

Per2 Cys215,

Cys841 (human) (CP motif)

Cys956 (mouse) (CP motif)

Heme Proteins with the Thiolate Axial Ligand

13

Axial ligands Abs. (nm) Function

Fe(III) Fe(II) Fe(II)-CO Fe(II)-CO

P450 Cys/ Cys/ Cys/CO 450 Monooxygenation

NOS Cys/ Cys/ Cys/CO 450 Monooxygenation

CPO Cys/Glu Cys/ Cys/CO 443 Halogenation

APO Cys/Glu Cys/ Cys/CO 450 Peroxygenation

SoxAX Cys/His Thiosulfate oxida.

CBS Cys/His Cys/His His/CO 420 Cystathione synth.

CooA Cys/Pro His/Pro His/CO 420 Transcription

Nitroph. Cys/ NO transfer

BxRcoM Cys/His Met/His His/CO 420 Transcription

P450, NOS, CPO Heme sensor: CP motif, heme-regulatory motif (HRI)

14

Fe(III)

S–

N

HN

His

Fe(II)

N

HN

His

Fe(II)

N

HN

His

e– CO

C

O

NH

O

N

OO

N

NH

His

Cys–Pro

CP motif (HRM: heme-regulatory motif)

Heme redox-dependent ligand switch

420 nm

15

16 16

HRI - eIF2 kinase (Translation regulation) IRP2 – Regulator of iron metabolism

Hap1 - Transcriptional activator Irr - Translation inhibitor

Bach1 - Transcriptional repressor

• Heme regulatory motif in hemeproteins mediates

the regulation of their function by heme.

Heme Regulatory Motif (CP motif) in

HRI and Other Proteins

Fast Heme Dissociation

17

18

1 138 619

kinase domain (KD) HRI NTD

Cys

His

Cys

His

heme

Inactive Active

heme

eIF2

ATP ADP

eIF2 eIF2 P

Association/dissociation of heme iron regulates the kinase

reaction associated with protein synthesis.

Specific features of the heme-responsive heme sensor proteins

1. Thiolate as the heme-binding (heme-sensing) site.

2. CP (Cys-Pro) motif or HRM (heme-regulatory motif).

3. Fast heme dissociation rate.

4. Redox-dependent heme binding.

5. Protein flexibility and plasticity.

Handbook of Porphyrin Science, Vol. 15, World Scientific,

pp. 399-460 (2011).

19

20

Heme-regulated eukaryotic initiation factor 2 (eIF2) kinase Heme-regulated inhibitor (HRI)

Cys409 Pro410

His119 His120

P-Tyr193

P-eIF2α Thr485-P

Thr490-P

P P

P P

P P

P P

P P

P P P P

P P

P P

P P

P P

P P

P P

P P P P

P P N

C

NTD KD

N-lobe

C-lobe

(1) Heme dissociation (2) Auto-phosphorylation (3) Multiple phosphorylation (4) eIF2α phosphorylation (1)

(2)

(3)

(4)

Igarashi et al. J. Biol. Chem. 279, 15752 (2004).

Miksanova et al. Biochemistry 45, 9894 (2006).

Martinkova et al. FEBS Lett. 581, 4109 (2007).

Igarashi et al. J. Biol. Chem. 283, 18782 (2008).

Mukai et al. Protein Pept. Lett. 18, 1251 (2011).

Igarashi et al. FEBS J. 278, 918 (2011).

22

1 138 619

kinase domain (KD) HRI NTD

Cys

His

Cys

His

heme

Inactive Active

heme

eIF2

ATP ADP

eIF2 eIF2 P

Association/dissociation of heme iron regulates the kinase

reaction associated with protein synthesis.

Specific features of the heme-responsive heme sensor proteins

1. Thiolate as the heme-binding (heme-sensing) site.

2. CP (Cys-Pro) motif or HRM (heme-regulatory motif).

3. Fast heme dissociation rate.

4. Redox-dependent heme binding.

5. Protein flexibility and plasticity.

Handbook of Porphyrin Science, Vol. 15, World Scientific,

pp. 399-460 (2011).

23

24

N

NN

NFe

COOH

COOH

HRI

Cys409

His119/120

N-terminal

C-terminal

Iron Metabolism Regulates p53 Signaling through Direct Heme-p53 Interaction and Modulation of p53 Localization, Stability, and Function Cell Reports 7, 180 (2014) Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy

26

Tumor Suppressor p53 Protein Is Downregulated during Iron Excess Hemin Directly Modulates p53 Stability without Involving the Transcription Factor Hypoxia-Inducible Factor 1

Tumor Suppressor p53 Protein Directly Binds to Heme In Vitro

The C-Terminal HRM in p53 Protein Is Required for Heme Binding

Heme Interferes with p53-DNA Interaction In Vitro and In Vivo Heme Destabilizes p53 Protein Mainly through the Ub-Proteasome System Heme Triggers Nuclear Export of p53 Protein Involving Its C-Terminal Nuclear Export Sequence Iron Deprivation Suppresses Growth and Tumorigenicity of Human Colon Carcinoma Cells in a p53-Depedent Manner

27

Figure 3

Cell Reports 2014 7, 180-193DOI: (10.1016/j.celrep.2014.02.042)

Copyright © 2014 The Authors Terms and Conditions

28

Discovery of a Heme-sensing Protein That Regulates Expression of Iron Acquisition Genes in a Bacterial Pathogen: A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus J. Biol. Chem. 291, 29 (2016) The bacterial pathogen Staphylococcus aureus causes a number of infectious diseases, including meningitis and osteomyelitis. It is a common cause of blood-borne infections in North America. The pathogen has several mechanisms for iron uptake during times of iron scarcity. In this describe their discovery of a DNA-binding regulatory protein that fine-tunes the expression of the iron-regulated genes. The protein, called SbnI, senses heme to control the expression of genes involved in iron uptake. When bound to heme, SbnI doesn’t bind to its DNA promoter and turn on the expression of proteins needed for iron acquisition through the siderophore staphyloferrin B. This is the first study to investigate the importance of SbnI in siderophore synthesis, and our data clearly implicate a role for SbnI in transcription control from regions within the sbn operon.

29

FIGURE 12. Proposed model of SbnI-mediated gene regulation in response to heme. In conditions of low iron and low heme, SbnI dimer binds to DNA within the sbnC coding region and promotes expression of genes sbnD–H. Under conditions of low iron and high heme, heme binds to SbnI, preventing binding of SbnI to DNA and thereby resulting in decreased expression of genes sbnD–H.

30

Discovery of Intracellular Heme-binding Protein HrtR, Which Controls Heme Efflux by the Conserved HrtB-HrtA Transporter in Lactococcus lactis J. Biol. Chem. 287, 4752 (2012) Background: Heme is an essential cofactor yet toxic in free form, necessitating strict intracellular control. Results: A heme sensor regulates the conserved hrtBA genes in Lactococcus lactis, whose products mediate heme efflux. Conclusion: L. lactis controls heme homeostasis by sensing intracellular heme and activating heme efflux. Significance: The use of an intracellular heme sensor to control heme efflux

constitutes a novel paradigm for bacterial heme homeostasis. Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor

to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis. 31

FIGURE 6. Schematic representation showing two distinct bacterial heme-sensing systems, one extracellular and one intracellular, that regulate heme homeostasis by HrtBA-mediated efflux. Left panel, extracellular heme-sensing induces the two component HssSR system. Once stimulated, HssR activates hrtBA expression, as reported (25). Right panel, inL. lactis,hemeis taken up by fhuDBA gene products (green ovals) (35).4 Internalizedhemebinds to available HrtR protein to relieve repression of the hrtRBA operon. Activation of hrtBA results in heme efflux. Red asterisk, heme. Blue lightning represents sensor activation.

32

Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator Sci. Rep. 6:18703 (2016) DOI: 10.1038/srep18703 The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the “active site conversion” from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel “two-step self-oxidative modification” mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.

33

Figure 8. A schematic representation of the mechanism for the oxidative modification of Irr. H2O2 generation (upper scheme) and H2O2 activation steps (lower scheme).

34

Heme impairs the ball-and-chain inactivation of potassium channels Proc. Nat. Acad. Sci. USA E4036 (2013) DOI/10.1073/pnas.1313247110. ABSTRACT: Fine-tuned regulation of K+ channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K+ channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K+ channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K+ current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore. Signficance: Heme, traditionally viewed as a stable protein cofactor such as in hemoglobin, also serves as an acute signaling molecule and is cytotoxic at high concentrations. Here, we show that free intracellular heme potently enhances A-type potassium channel function. Such channels determine action potential frequency in excitable cells, and their dysfunction often contributes to pathological hyperexcitability, such as in pain and epilepsy. Binding of free heme at nanomolar concentrations to the “balland-chain” N terminus of A-type potassium channels, which typically closes the channels, introduces a stable structure in the otherwise disordered region and allows for a greater efflux of potassium ions, thus reducing cellular excitability. Heme therefore could be a powerful negative-feedback regulator in brain and muscle function.

35

Fig. 2. Mutagenesis of N-terminal inactivation domain. (A) Schematic presentation of the Kv1.4 protein comprising the cytosolic N and C termini, as well as the membrane-delimited 6TM domain (S1–S6). Residue numbers, based on rat Kv1.4, are indicated and the amino acid sequence corresponding to the red bar is shown below, highlighting C13, H16, and H35

36

37

A heme-binding domain controls regulation of ATP-dependent potassium channels Proc. Nat. Acad. Sci. USA 113, 3785 (2016) Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

38

Proposed redox regulatory mechanism of human BK channel and HO2 under hypoxic/normoxic condition. This figure describes a working hypothesis for how HO2 and the BK channel system may coordinately respond to intracellular redox changes, e.g. hypoxia and normoxia. Under hypoxic or more reducing conditions, the thiol/disulfide redox switches in HO2 and the BK channel would be in the dithiol states, with HO2 having low affinity and BK channel having high affinity for Fe3+-heme. The combined effects of low heme affinity and low O2 concentration would decrease HO2 activity, resulting in an increase in heme and decrease in CO concentrations. Increase in heme affinity of the BK channel, combined with high heme and low CO levels would promote closing of the channel, and depolarization of the cell membrane. Under more oxidizing and/or normoxic conditions, the redox active thiols of HO2 and the BK channel would exist in oxidized states, with HO2 having high affinity and the BK channel having low affinity for Fe3+-heme. Furthermore, the increased O2 levels would stimulate HO2 activity to generate CO, which would activate the channel, leading to hyperpolarization of the cell membrane.

Heme closes

channel.

?

39

Antioxid. Redox Signal 14, 1039 (2011). Model for redox regulation of HO2 and BK channel activity. The membrane-spanning, HBD, RCK1, RCK2, and C-terminal domains of the BK channel (one of the four pore-forming α subunits is removed to show the internal channel) are shown in interaction with HO2, which contains a core catalytic domain that binds and metabolizes heme and a C-terminal regulatory region that contains the thiol/disulfide switch. The surface representation of HO2 is based on its crystal structure, which lacks the redox switch, here represented by two rectangles (9a).The cartoon of the BK channel is loosely derived from its EM structure (61a). Under normoxic conditions, the channel opens because inhibitor heme has dissociated from the HBD (due to its low heme affinity in the SS state) or because CO (generated by HO2) is bound??. Under hypoxic conditions, the channel is closed because inhibitor heme is bound (due to the high affinity of HBD for heme in its reduced RSH state) and because CO levels are relatively low (due to low affinity of HO2 for heme and to low O2 levels). Thus, heme is bound to HO2 under normoxic conditions and to the HBD of the BK channel under hypoxia. HBD, heme-binding domain; RCK, regulator of conductance of potassium.

?

40

41

DGCR8 Heme is involved in microRNA processing Nat. Struc. Mol. Biol. 14, 23 (2007). DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-liganded Heme Protein J. Biol. Chem. 286, 16716 (2011). Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing Proc. Nat. Acad. Sci., USA 109, 1919 (2012). Processing of microRNA primary transcripts requires heme in mammalian cells Proc. Nat. Acad. Sci., USA 111, 1861 (2014) The DGCR8 RNA-Binding Heme Domain Recognizes Primary MicroRNAs by Clamping the Hairpin Cell Rep. 7, 1994 (2014).

Figure 6

Cell Reports 2014 7, 1994-2005DOI: (10.1016/j.celrep.2014.05.013)

Copyright © 2014 The Authors Terms and Conditions

Models of How a pri-miRNA Is Recognized by the Microprocessor (A) The basal junction anchoring model (Han et al., 2006). (B) The apical junction anchoring model (Zeng et al., 2005). (C) Our proposed molecular clamp model. See Discussion for details. The

DGCR8 subunits in a dimer are shown in red and cyan. The thick avocado strands represent 5′ and 3′ mature miRNAs.

Cell Reports 2014 7, 1994-2005DOI: (10.1016/j.celrep.2014.05.013)

Copyright © 2014 The Authors Terms and Conditions

The DGCR8 RNA-Binding Heme Domain Recognizes Primary MicroRNAs by Clamping the Hairpin Quick-Cleveland, J. et al. Cell Rep. 7, 1994 (2014) ....... in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin......

44

Highlights •A unique RNA-binding domain is uncovered in the pri-miRNA processing factor DGCR8 •The RNA-binding heme domain (Rhed) binds the ends of the pri-miRNA hairpin •The Rhed and its RNA-binding surface are important for pri-miRNA processing •The molecular clamp model explains recognition of all pri-miRNA structural features Summary Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ∼30 bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here, we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds

PpsR, a Regulator of Heme and Bacteriochlorophyll Biosynthesis, Is a Heme-sensing Protein J. Biol. Chem. 287, 13850 (2012) Background: PpsR controls synthesis of heme and bacteriochlorophyll in purple photosynthetic bacteria. Results: PpsR binds heme as a co-factor and changes its DNA binding pattern. Conclusion: Heme affects the ability of PpsR to regulate tetrapyrrole gene expression. Significance: A cysteine axial ligand to heme is in the helix-turn-helix domain providing a way for heme to affect DNA binding properties of PpsR Heme-mediated regulation, presented in many biological processes, is achieved in part with proteins containing heme regulatory motif. In this study, we demonstrate that FLAGtagged PpsR isolated from Rhodobacter sphaeroides cells contains bound heme. In vitro heme binding studies with tagless apo-PpsR show that PpsR binds heme at a near one-to-one ratio with a micromolar binding constant. Mutational and spectral assays suggest that both the second Per-Arnt-Sim (PAS) and DNA binding domains of PpsR are involved in the heme binding. Furthermore, we show that heme changes the DNA binding patterns of PpsR and induces different responses of photosystem genes expression. Thus, PpsR functions as both a redox and heme sensor to coordinate the amount of heme, bacteriochlorophyll, and photosystem apoprotein synthesis thereby providing fine tune control to avoid excess free tetrapyrrole accumulation

47

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Involvement of Heme Regulatory Motif in Heme-Mediated Ubiquitination and Degradation of IRP2 Mol. Cell 19, 171 (2005) Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

49

Human Tryptophanyl-tRNA Synthetase Binds with Heme To Enhance Its Aminoacylation Activity Biochemistry 46, 11291 (2007) ABSTRACT: Mammalian tryptophanyl-tRNA synthetases (TrpRSs) are Zn2+-binding proteins that catalyze the aminoacylation of tRNATrp. The cellular expression level of human TrpRS is highly upregulated by interferon-g (IFN-g). In this study, a heme biosynthesis inhibitor, succinylacetone (SA), was found to inhibit cellular TrpRS activity in IFN-g-activated cells without affecting TrpRS protein expression. In addition, supplementation of lysates from the SA-treated cells with hemin fully restored TrpRS activity to control levels. Biochemical analyses using purified TrpRS demonstrated that heme can interact strongly with Zn2+-depleted human full-length TrpRS with a stoichiometric heme:protein ratio of 1:1 to enhance the aminoacylation activity significantly. In contrast, the Zn2+-bound form of TrpRS did not bind heme. Further studies using site-directed mutagenesis clarified that the Zn2+-unbound human H130R mutant cannot bind heme. These results provide the first evidence of the involvement of heme in regulation of TrpRS aminoacylation activity. The regulation mechanism and its physiological roles are discussed.

50

51

Heme iron complex is the active center, but not associated with O2 and electron transfer

Regulation of Aldoxime Dehydratase Activity by Redox-dependent Change in the Coordination Structure of the Aldoxime-Heme Complex J. Biol. Chem. 280, 5486 (2005) Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, α, and β bands at 415, 555, and 524 nm, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (∼3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.

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53

Control of carotenoid biosynthesis through a heme-based cis-trans isomerase Nature Chem. Biol. 11, 598 (2015) Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-z-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15′ carbon-carbon double bond in 9,15,9′-cis-z-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b–dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

54

Figure 1: Z-ISO is an isomerase and integral membrane protein localized to chloroplasts. 55

56

Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome ACS Chem. Biol. 11, 882 (2016) ABSTRACT: The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets

57

Figure 4. Ingenuity Pathway Analysis (IPA) bioinformatics analysis of artemisinin binding proteins and proposed binding mode. (A) Cellular localization of artemisinin-binding proteins. (B) Functional classification of artemisinin-binding proteins. (C) Top 5 molecular and biological functions to which artemisinin-binding proteins are classified. (D) Binding mode of heme-activated artemisinin with proteins. (E) Binding mode of artemisinin with heme-containing or -bounded proteins.

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Circadian rhythms (Body clock)

Sleep

Feed Awake

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Molecular Mechanism of Input Systems (light) on mammalian

SCN

Glu

..

SCN neuron

Ca2+ Kinase activation

CREB phosphorylation

mPer1 expression

Ocsillation synchronizes

with the environment

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CL

OC

K

BM

AL

1

per E-box

cry E-box

CL

OC

K

BM

AL

1

PER

CRY

PER

CRY (-)

(-)

degradation Output

Rhythm

nucleus

cytoplasm

Time / h

Expre

ssio

n o

f P

ER

0 6 12 24

100

50

0

Oscillation of PER

E-BOX 5’ CACGTG 3’

61

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Post-translational modifications in the circadian clock. The current understanding for post-

translational regulation of the circadian clock is modeled. A) Clock and Bmal1 dimerize and

transcriptionally activate clock-controlled genes, including Per and Cry. B) Per and Cry interact in the

cytosol. C) Per is phosphorylated by CK1δ/ε, causing it to translocate into the nucleus. D) Per and

Cry repress Clock and Bmal1 activity. E) Phosphorylated Per1 is potentially dephosphorylated by

protein phosphatase 1 (PP1), either in the cytosol or in the nucleus (causing nuclear export). P,

phosphorylation. Dotted lines indicate hypothetical mechanisms.

FESEB J. 26, 3602 (2012)

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The core oscillator of the mammalian circadian clock. Science 337, 165 (2012)

(A) The transcriptional-translational feedback loop that makes up the core oscillator of the mammalian circadian

clock. CLOCK and BMAL1 bind to E-box DNA sequences to activate clock-controlled genes (cogs), including

those that code for the oscillator proteins PER and CRY. PER and CRY are translated in the cytoplasm and then

cycle back into the nucleus to directly repress CLOCK:BMAL1. As PER and CRY levels drop, CLOCK:BMAL1

reactivates another round of transcription. (B) The CLOCK (green) and BMAL1 (blue) proteins heterodimerize to

bind DNA through their N-terminal bHLH domains. The structure reported by Huang et al. shows that the PAS A

domains dimerize symmetrically; in contrast, the PAS B domains form a head-to-tail interaction, mediated by a

conserved Trp residue on BMAL1 that binds into CLOCK PAS B. The analogous Trp on CLOCK projects into

solvent for putative interactions with CRY. To inhibit transcription, the PER tandem PAS domains may interact

with those of CLOCK:BMAL1. Unstructured regions of CLOCK and BMAL1 (dotted lines) are important for

transcriptional activation and histone acetyltransferase (HAT) activity. Gln, glutamine; ccgs, clock-controlled

gene sequences.

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Crystal structure of the heterodimeric CLOCK:BMAL1 Transcriptional activator complex

Science 337, 189 (2012)

Mitchell Lazar et al. Science 318, 1786 (2007).

Fraydoon Restinejad et al. Nature Stuc. Mol. Biol. 14, 1207 (2007).

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66

Nuclear receptor Rev-erb: up, down, and all around

Trends in Endocrinology and Metabolism 25 (11), 586 (2014)

Rev-erb is a nuclear receptor that links circadian rhythms to

transcriptional control of metabolic path-ways. Rev-erb is a potent

transcriptional repressor and plays an important role in the core

mammalian molecular clock while also serving as a key regulator of

clock output in metabolic tissues including liver and brown adipose

tissue (BAT). Recent findings have shed new light on the role of

Rev-erb and its paralog Rev-erbb in rhythm generation, as well as

additional regulatory roles for Rev-erb in other tissues that

contribute to energy expenditure, inflammation, and behavior. This

review highlights physiological functions of Rev-erb and b in

multiple tissues and discusses the therapeutic potential and

challenges of targeting these pathways in human disease

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Figure 1. Binding configurations of

Rev-erb. (A) At single RORE

[retinoic acid receptor-related

orphan receptor (ROR) element]

sites, Rev-erb can inhibit

transcription passively by competing

for binding with ROR proteins. (B)

Two Rev-erb proteins bound

independently to separate RORE

motifs can recruit nuclear

corepressor 1 (NCoR) in a heme-

dependent manner. The NCoR

complex recruits histone

deacetylase 3 (HDAC3), which

deacetylates surrounding histone

tails and represses target gene

transcription. (C) A Rev-erb dimer

bound to a RevDR2 (Rev-erb direct

repeat separated by 2bp) motif can

also recruit NCoR, with similar

regulatory effects as in (B).

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Figure 2

Tissue-specific

rhythms and

functions of Rev-

erbα. Diagram of

Rev-erbα

circadian

expression

patterns and

regulatory targets,

in various tissues.

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Figure 3. REV-ERBs Can Assume at Least Six Different Heme Binding States PLoS Biol. 7, e1000043 (2009)

In the absence of heme, the ligand pocket of REV-ERBs is stabilized with the side chains of core hydrophobic

residues. Heme can bind to the REV-ERBs in either reduced Fe(II) or oxidized Fe(III) states, and while reduced

is in a five or six coordinate state. REV-ERBs also bind the diatomic gases CO and NO through the creation of a

coordinate bond between the Fe(II) center of heme.

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High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear

Receptor Rev-erbβ Leads to its Degradation and Indirectly Regulates its

Interaction with Nuclear Receptor Corepressor

J. Biol. Chem. in press (2016)

Abstract Rev-erbα and Rev-erbβ are heme-binding nuclear receptors (NRs) that repress the

transcription of genes involved in regulating metabolism, inflammation and the circadian

clock. Previous gene expression and co-immunoprecipitation studies led to a model in

which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an

active repressor complex. However, in contradiction, biochemical and crystallographic

studies have shown that heme decreases affinity of the ligand-binding domain (LBD) of

Rev-erbs for NCoR1 peptides. One explanation for this discrepancy is that the LBD and

NCoR1 peptides used for in vitro studies cannot replicate key features of the full-length

proteins used in cellular studies. However, combined in vitro and cellular results

described here demonstrate that heme does not directly promote interactions between

full-length Rev-erbβ (FLRev-erbβ) and an NCoR1 construct encompassing all three NR

interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbβ:DNA

complexes; furthermore, heme, at high concentrations,destabilizes the FLRev-erbβ-

NCoR1 complex. The interaction between FLRev-erbβ and NCoR1 as well as Rev-erbβ

repression at the Bmal1 promoter appear to be modulated by another cellular factor(s),

at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest

that heme is involved in regulating degradation of Rev-erbβ in a manner consistent with

its role in circadian rhythms maintainance. Finally, the very slow rate constant (10-6 s-1)

for heme dissociation from Rev-erbb, rules out a prior proposal that Rev-erbb acts as

Intracellular heme sensor.