An Escherichia coli expression–based method for heme substitution

Woodward, Joshua J.; Martin, Nathaniel I.; Marletta, Michael A.

doi:10.1038/nmeth984

Cited by 63 publications

(98 citation statements)

References 14 publications

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“…First, expression was carried out in the presence of added ␦-aminolevulinic acid, a heme precursor commonly used to enhance the abundance of heme inside expressed heme proteins. Second, an E. coli protein expression strain capable of taking up exogenously added heme was used to express the protein in the presence of heme-supplemented growth media (25). Although both methods increased the fraction of as-isolated holo-HemQ, the magnitude of the increase was marginal.…”

Section: Methodsmentioning

confidence: 99%

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

Mayfield

Hammer

Kurker

et al. 2013

Journal of Biological Chemistry

112

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Section: Methodsmentioning

confidence: 99%

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

Mayfield

Hammer

Kurker

et al. 2013

Journal of Biological Chemistry

112

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“…Excess hemin was then removed by applying the sample to a PD-10 column equilibrated with buffer A for sGC ␣1␤1 I145Y/I149Q or buffer B (50 mM Hepes, pH 7.4, 50 mM NaCl, 5 mM DTT) for sGC ␣1␤1 F74Y. Heme stoichiometry was determined as described previously (30). These proteins were characterized both as isolated and after heme reconstitution to ensure the procedure did not affect protein activity or ligand binding characteristics.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Derbyshire

Deng

Marletta

2010

Journal of Biological Chemistry

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Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC).The heme cofactor of ␣1␤1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain ␤1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in fulllength sGC we mutated several conserved distal heme pocket residues (␤1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat ␤1 distal heme pocket (Ile-145 3 Tyr and Ile-149 3 Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe II -O 2 complexes and is likely the first observation of a Fe II -O 2 complex in the full-length ␣1␤1 protein. Additionally, these data suggest that atypical sGCs stabilize O 2 binding by a hydrogen bonding network involving tyrosine and glutamine.Soluble guanylate cyclase (sGC) 3 is the most thoroughly characterized receptor for the gaseous signaling agent nitric oxide (NO). NO induced activation of sGC is critical to several physiological processes, including neurotransmission, vasodilation, and platelet aggregation (1-3). The importance of sGC to physiological function has been clearly demonstrated over the last decade (4, 5); however, much less is understood about the molecular mechanisms that regulate enzyme activity.sGC is a heterodimeric hemoprotein consisting of two homologous subunits, ␣ and ␤. The ␣1␤1 heterodimer is the most prevalent and commonly studied protein. Despite the same histidine ligated heme and iron oxidation state as found in the globins, sGC shows no measurable affinity for O 2 and therefore can selectively bind NO in the presence of O 2 (reviewed in Refs. 6, 7). Interestingly, not only does sGC discriminate against O 2 binding to the heme, but both the Fe II -unligated and Fe II -NO species are stable in an aerobic environment (8). This is in stark contrast to other hemoproteins that readily bind and ultimately react with O 2 (9 -12). The mechanism by which sGC discriminates against O 2 binding as well as the molecular events that lead to activation remain to be elucidated.There have been several proposals on the mechanism of ligand discr...

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“…The His 6-tag was cleaved using the TEV protease. Quantification of heme cofactor was performed by absorption spectroscopy after protein denaturation and by HPLC (37). The Fe(III)-imidazole complex was calculated to have 429 ϭ 75 Ϯ 2 mM Ϫ1 cm Ϫ1 by the first method and 429 ϭ 87 Ϯ 1 mM Ϫ1 cm Ϫ1 by the second.…”

Section: Materials See Si Text For Detailsmentioning

confidence: 99%

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

Agapie

Suseno²,

Woodward³

et al. 2009

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

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The role of nitric oxide (NO) in the host response to infection and in cellular signaling is well established. Enzymatic synthesis of NO is catalyzed by the nitric oxide synthases (NOSs), which convert Arg into NO and citrulline using co-substrates O2 and NADPH. Mammalian NOS contains a flavin reductase domain (FAD and FMN) and a catalytic heme oxygenase domain (P450-type heme and tetrahydrobiopterin). Bacterial NOSs, while much less studied, were previously identified as only containing the heme oxygenase domain of the more complex mammalian NOSs. We report here on the characterization of a NOS from Sorangium cellulosum (both fulllength, scNOS, and oxygenase domain, scNOSox). scNOS contains a catalytic, oxygenase domain similar to those found in the mammalian NOS and in other bacteria. Unlike the other bacterial NOSs reported to date, however, this protein contains a fused reductase domain. The scNOS reductase domain is unique for the entire NOS family because it utilizes a 2Fe2S cluster for electron transfer. scNOS catalytically produces NO and citrulline in the presence of either tetrahydrobiopterin or tetrahydrofolate. These results establish a bacterial electron transfer pathway used for biological NO synthesis as well as a unique flexibility in using different tetrahydropterin cofactors for this reaction.heme protein ͉ iron-sulfur cluster ͉ reductase ͉ tetrahydrobiopterin ͉ tetrahydrofolate N itric oxide (NO) is recognized as an important signaling molecule as well as a cytotoxin (1-3). A number of diseases are intimately tied to improper function of NO in humans (1-3). Given the importance of NO to human health, a wealth of studies has been performed to address questions regarding NO synthesis and regulation (4-9). Isolation and structural characterization of the proteins responsible for NO synthesis have led to important developments in understanding the molecular processes of NO formation (10-12). Three isoforms of nitric oxide synthase (NOS), iNOS, eNOS, and nNOS, have been characterized in mammals. These enzymes contain an oxygenase domain where the catalysis takes place and a reductase domain that is involved in electron transfer (4, 9). NOS is functional only as a homodimer, and requires the binding of a Ca 2ϩ -calmodulin (CaM) complex for electron transfer between the reductase and oxygenase domains. The reductase domain transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) to the P450-type heme in the oxygenase domain (13). NOS contains an additional reduced pterin cofactor in the oxygenase domain [H 4 B, (6R)-tetrahydro-l-biopterin], which is required for NO formation and is involved in electron transfer processes during catalysis at the heme (14-16).NOS catalyzes the conversion of Arg into NO and citrulline using O 2 and NADPH involving two catalytic steps. In the first reaction, Arg is oxidized to N G -hydroxy-arginine (NHA). Nitric oxide is generated in the second half of the cycle, with the conversion...

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An Escherichia coli expression–based method for heme substitution

Cited by 63 publications

References 14 publications

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

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