Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Varfaj, Fatbardha; Lampe, Jed N.; Montellano, Paul R. Ortiz de

doi:10.1074/jbc.m112.378042

Cited by 27 publications

(36 citation statements)

References 31 publications

(35 reference statements)

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“…Moreover, an NMR study of different single and double mutants of the heme oxygenase HO-2 involved in signaling and regulatory processes demonstrates that, analogous to our observations, the redox state of a given cysteine out of the three cysteines in HO-2 is sensitive to the absence of one or both cysteines (49).…”

mentioning

confidence: 69%

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Botello-Morte

Pellicer

Sein‐Echaluce

et al. 2016

Antioxidants & Redox Signaling

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Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. , suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C 101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 24, 173-185.

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mentioning

confidence: 69%

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Botello-Morte

Pellicer

Sein‐Echaluce

et al. 2016

Antioxidants & Redox Signaling

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“…These proteins have high affinity for heme, usually less than pM for the globins (61) and in the nM range for heme oxygenase (62,63). The remaining portion is in the form of a regulatory heme pool, which is available for trafficking around the cell by as yet unspecified mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Regulation of intracellular heme trafficking revealed by subcellular reporters

Yuan

Rietzschel

Kwon

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

108

126

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Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditiselegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

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“…However, no change in activity or heme binding occurred when CP motif cysteines were mutated to Ala (31), and His-45 ligates the substrate heme for oxidation (30). In normoxia, a disulfide forms between Cys-265 and Cys-282 of two HRMs, with a small increase in active site heme affinity (32). Disulfide reduction to dithiol may lower active site heme affinity and increase the cellular heme pool.…”

Section: Heme Homeostasismentioning

confidence: 99%

“…Disulfide reduction to dithiol may lower active site heme affinity and increase the cellular heme pool. CP motifs may thus be nonessential for HO-2 heme binding and instead have signaling or molecular interaction functions (31)(32)(33).…”

Section: Heme Homeostasismentioning

confidence: 99%

Heme Sensor Proteins

Girvan

Munro

2013

Journal of Biological Chemistry

128

116

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Heme is a prosthetic group best known for roles in oxygen transport, oxidative catalysis, and respiratory electron transport. Recent years have seen the roles of heme extended to sensors of gases such as O 2 and NO and cell redox state, and as mediators of cellular responses to changes in intracellular levels of these gases. The importance of heme is further evident from identification of proteins that bind heme reversibly, using it as a signal, e.g. to regulate gene expression in circadian rhythm pathways and control heme synthesis itself. In this minireview, we explore the current knowledge of the diverse roles of heme sensor proteins. Heme-responsive Proteins: Defining FeaturesHeme homeostasis is crucial. Free heme at Ͼ1 M is cytotoxic, mainly by producing reactive oxygen species. Iron intake accounts for only a small proportion of mammalian requirements, so iron recycling (particularly from heme) is critical (1). Tight regulation of heme synthesis/breakdown is needed. Heme regulates its own fate and controls several other biological processes. Heme-responsive proteins elicit cellular responses by binding/debinding heme or via changes in heme ligation (e.g. by gases) or redox state. They can be divided into two classes: nuclear receptor (NR) 2 hemoproteins and heme sensors with no NR function. Each heme-responsive protein has a heme regulatory motif (HRM), usually containing a CP motif (2). The Cys residue of the CP motif is an axial heme ligand. No other residues are conserved across the protein class. The wider relevance of the CP motif is unclear because other hemoproteins (e.g. prostaglandin E 2 synthase, chloroperoxidase, and some cytochromes P450) also have a CP motif. The properties of members of the two main classes are described below. NR HemoproteinsSeveral heme-binding proteins occur in the NR superfamily, which is the largest transcription factor superfamily (summarized in Table 1). NRs bind specific DNA motifs in response to small molecule signaling. They generally share conserved domain architecture, with a DNA-binding region containing two zinc fingers, a ligand-binding domain, and activation domains (3). Usually, ligand binding induces conformational changes that dissociate partner proteins, allowing DNA binding and/or changes to dimerization status or cellular localization that enable the protein to elicit a response. A subfamily of NRs binds heme at their ligand-binding domain and so act as heme sensors, as discussed below. Circadian Rhythm Heme SensorsCircadian rhythms are regulated by feedback loops at transcriptional/translational levels. Heme is critical in this process (4). In vertebrates, the expression of several day/night cycle genes, as well as Rev-erb␣, is controlled by binding a heterodimer of Clock (or NPAS2 (neuronal PAS domain protein 2)) and Bmal1 to their 5Ј-UTR, thus switching on transcription. Expression of Bmal1 and NPAS2/Clock is repressed in turn by binding of Rev-erb␣ to a homodimeric partner (5). Rev-erb␣ and homologs (Rev-erb␤ and E75, with the latter involved in inse...

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Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Cited by 27 publications

References 31 publications

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

Regulation of intracellular heme trafficking revealed by subcellular reporters

Heme Sensor Proteins

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