Resonance Raman Studies Indicate a Unique Heme Active Site in Prostaglandin H Synthase

Lou, Bih‐Show; Snyder, Jennifer K.; Marshall, Paul; Wang, Jinn‐Shyan; Wu, Gang; Kulmacz, Richard J.; Tsai, Ah‐Lim; Wang, Jianling

doi:10.1021/bi001257c

Cited by 32 publications

(43 citation statements)

References 61 publications

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“…3Aa). This Raman spectrum was quite different from those reported for PGHS-1 (29,40). In PGHS-1, two Fe-C stretching modes were observed at 502 and 529 (40) and at 496 A small shoulder appeared at 524 cm Ϫ1 for PGHS-2.…”

Section: Resultscontrasting

confidence: 78%

“…Previously, Marnett and co-workers (28) reported that the mutation of Gln-189 to Val showed a drastic decrease in peroxidase activity by 2 orders of magnitude and also a loss of cyclooxygenase activity, proposing that the neutral amide group of Gln-189 is responsible for the peroxidase reaction in PGHS-2. However, a recent resonance Raman study for the CO adduct of PGHS-1 suggested that no hydrogen bond is formed between ironbound CO and the surrounding amino acid residues (29). In addition, the x-ray structure of PGHS-2 revealed that the amide group in Gln-189 is located relatively far from the heme iron (6.26 Å) (7) compared with the guanidinium group of Arg in HRP (4.61 Å) (30).…”

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confidence: 99%

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Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

Ichimura

Uchida

Taniguchi

et al. 2007

Journal of Biological Chemistry

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Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H 2 , but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln3 Arg), Asn (Gln3 Asn), and Val (Gln3 Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a sixcoordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.Prostaglandin endoperoxide H synthase (PGHS, 3 also known as cyclooxygenase or COX) is a membrane-bound heme-containing protein catalyzing the first committed step in prostanoid biosynthesis (1-3) including two sequential enzymatic reactions, bis-oxygenation of arachidonic acid to prostaglandin G 2 (PGG 2 ) (cyclooxygenase reaction) and the reduction of PGG 2 to prostaglandin H 2 (PGH 2 ) (peroxidase reaction). PGH 2 is, then, further metabolized to various kinds of prostaglandins, prostacyclins, and thromboxanes by the appropriate synthase (2). Two isoforms of PGHS have been discovered thus far, PGHS-1 and PGHS-2, both of which are composed of ϳ600 amino acid residues, sharing more than 60% sequence identity (4, 5), and their crystal structures are essentially superimposable (6 -8).Although both the PGHS isozymes function as a homodimer of ϳ70-kDa subunits and have similar catalytic properties, they have distinctly different biological functions. PGHS-1 is a "housekeeping" enzyme, expressed constitutively in most tissues, and produces prostaglandins to regulate cellular responses to hormonal stimulation and to regulate vascular homeostasis. PGHS-2, in contrast, is inducibly expressed i...

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Section: Resultscontrasting

confidence: 78%

mentioning

confidence: 99%

Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

Ichimura

Uchida

Taniguchi

et al. 2007

Journal of Biological Chemistry

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“…These values are typical of hexa-coordinated low-spin (LS) ferric heme proteins. 27 The RR spectrum of ferrous deoxygenated GsGCS 162 has clear marker lines at 1357 cm -1 (ν 4 ), 1469 cm -1 (ν 3 ), and 1554 cm -1 (ν 2 ) (Fig. 5a, trace b), typical for a penta-coordinated high-spin (HS) ferrous state.…”

Section: Resonance Raman Spectroscopymentioning

confidence: 99%

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Pesce¹,

Thijs

Nardini

et al. 2009

Journal of Molecular Biology

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⁎Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS 162 ) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS 162 as an example of bishistidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS 162 is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS 162 is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS 162 is a penta-coordinated species, where the proximal HisF8-Fe bond is severed. Abbreviations used: GCS, globin-coupled sensor; GsGCS 162 , globin domain of GCS from Geobacter sulfurreducens; PAS, an acronym formed from the names of the first three proteins (the Period clock protein of Drosophila, the Aryl hydrocarbon receptor nuclear translocator of vertebrates, and the Single-minded protein of Drosophila) recognized as sharing such sensor motif; CooA, transcriptional activator of coo operons encoding proteins required to metabolize CO as a sole energy

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“…For heme-containing oxygenases and peroxidases, the "push-pull" mechanism has been proposed where the generation of Cpd I is facilitated by an anionic proximal ligand and/or positive polar distal residues (51). In peroxidases like horseradish peroxidase or cytochorme c peroxidase, the "push" effect is provided by the proximal histidine and "pull" effect is mainly provided by the distal histidine that serves as a general acid-base catalyst to generate and stabilize the Cpd I (16,52). For P450cam, since it lacks the distal residue that could promote the heterolytic O-O bond cleavage, the strong "push" effect from the proximal thiolate axial ligand is proposed to be responsive for the O-O bond heterolysis.…”

Section: Abts Assaymentioning

confidence: 99%

“…The RR spectra of all the ferric protein samples displayed a strong oxidation state marker band ν 4 at around 1370 cm , respectively, indicating a five-coordinate (5C), high-spin (HS) heme iron (51)(52)(53). While for all other samples, the spectra displayed the ν 3 and ν 2 bands at around 1500 cm -1 and 1580 cm -1 , suggesting a six-coordinate (6C), low-spin (LS) heme iron component.…”

Section: Resonance Raman Spectroscopic Studymentioning

confidence: 99%

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Tian¹

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Resonance Raman Studies Indicate a Unique Heme Active Site in Prostaglandin H Synthase

Cited by 32 publications

References 61 publications

Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

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