Origin of the anomalous Soret spectra of carboxycytochrome P-450

Hanson, Louise Karle; Eaton, William A.; Sligar, Stephen G.; Gunsalus, I. C.; Gouterman, Martin; Connell, Charles R.

doi:10.1021/ja00425a050

Cited by 187 publications

(111 citation statements)

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“…The Soret band (350-450 nm, which arises from a π-π* transition of the heme) of the spectral intermediate of D251N P450 cam is considerably red-shifted compared to oxy-P450, and the spectrum of D251N oxy-P450 is the same as native oxy-P450. The Soret of the spectral intermediate is more like that of a p-type hyper spectrum than oxy-P450 and is similar to that of the ferrous carbon monoxide P450 cam complex (Gouterman, 1978;Hanson et al, 1976). Correlations among a wide range of metalloporphyrin spectra (Momenteau & Reed, 1994;Wang & Brinigar, 1979) suggest that red-shifting of both the Soret and visible bands is consistent with an increased electron density on the ligands of the spectral intermediate, compared to oxy-P450.…”

Section: Resultsmentioning

confidence: 83%

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

1997

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Cytochrome P450s are ubiquitous heme proteins responsible for various oxidative metabolic processes. The overall rate-determining step in the catalytic cycle of native cytochrome P450 cam is the reduction of the dioxygen complex, which has made detection of catalytic intermediates after this reduction impossible. However, for the site-specific mutant D251N cytochrome P450 cam (which affects proton transfer near the catalytic center), the overall rate-determining step occurs after the reduction of oxy-P450. As a consequence, we have observed in the UV-visible spectrum during catalytic turnover a new intermediate that is one electron reduced from oxy-P450 with an intact dioxygen bond.

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Section: Resultsmentioning

confidence: 83%

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

1997

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“…These include proximal thiolate protonation (12), loss or substitution of, for example, His, Met, or water for the Cys thiolate ligand (32,44), and lengthening of the thiolate Fe-S bond (18).…”

Section: Discussionmentioning

confidence: 99%

Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies

et al. 2002

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The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3 of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1 H-and 13 C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other wellcharacterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.

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“…Both the wavelength of the "Soret" peaks and the ratio of absorbances between the "450" band and the "370" band can be varied by changing solvent polarity. Splitting of a single Soret band into two bands ("hyper" type spectrum) observed in our model systems and in the CO-P-450 complex has been interpreted as a charge transfer from a mercptide sulfur orbital to the porphyrin eg (7r*) coupled to the normal porphyrin iX r* transition (25).…”

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confidence: 72%

Carbon monoxide binding to pentacoordinate mercaptide-heme complexes: kinetic study on models for cytochrome P-450.

Chang¹,

Dolphin²

1976

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

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Mercaptide anions form exclusively pentacoordinate heme complexes [R--hemeJ in polar and nonpolar solution over a wide range of mercaptide concentration. These complexes have a Soret peak at 408 nm and a formation constant of abut 2.5 X 104 M-', and combine with CO to give a CO cytochrome P450 type spectrum. Kinetics and cytochrome P450. The reaction of alkoxide anion with heme has also been examined but no evidence was found for the existence of the [RO--heme-CO] species. Cytochromes P-450 are a unique class of hemeproteins that catalyze the hydroxylation of a wide variety of organic compounds through the activation of molecular oxygen (1, 2). Although the primary locus of such enzyme systems is in the microsomal fraction of mammalian liver and adrenal cortex, much of our present knowledge concerning the properties of P-450 has been obtained from studies on the more readily available soluble P-450 of bacterial origin. A substantial amount of information is now available on the functional aspects of the catalytic reaction but comparatively little is known concerning the structure of the active site of the enzyme itself. Specifically, the nature and roles of axial ligands coordinated to the prosthetic group, protoheme, are yet to be elucidated. In the absence of definitive x-ray crystal structures of P-450, we have undertaken the approach of synthesizing model compounds which mimic P-450, in order that its mechanism of enzymic activities can be elucidated.The absorption spectrum with the unusual red-shifted Soret band of CO-P-450 complex has already been successfully duplicated with simple iron(II) porphyrins by having a mercaptide ion ligated trans to CO, at the fifth coordination site of heme (3-6). This evidence, coupled with earlier electron spin resonance experiments on both enzymes and model compounds (7)(8)(9), strongly implies that a mercaptide ligand is coordinated to all the ferric stages and at some stages of the ferrous complex in the P-450 catalytic cycle. In the absence of CO, our model exhibited a spectrum with absorption bands at wavelengths identical to those of reduced P-450 (3, 4). Since the unligated form of P-450 is assumed to be a pentacoordinate heme with subsequent ligation of CO or 02 taking place at the sixth site, we have examined the reaction between mercaptide ion and heme and studied the properties of the "mono" mercaptideheme species. We wish to report here the thermodynamics and kinetics of the interactions between mercaptide ion, heme, and CO, and to report some observations on the obligatory nature Abbreviations: P-450, cytochrome P-450; DMA, NN-dimethylacetamide; Me2SO, dimethyl sulfoxide; Heme, iron(II) protoporphyrin IX dimethyl ester; Hemin, iron(III) protoporphyrin IX chloride dimethyl ester.of the mercaptide ligand in bringing about the long-wavelength Soret peak of the CO complexes. MATERIALS AND METHODSProtohemin chloride dimethyl ester was prepared by insertion of iron into protoporphyrin IX dimethyl ester, which was synthesized from protoporphyrin via its ...

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Origin of the anomalous Soret spectra of carboxycytochrome P-450

Cited by 187 publications

References 1 publication

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies

Carbon monoxide binding to pentacoordinate mercaptide-heme complexes: kinetic study on models for cytochrome P-450.

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Origin of the anomalous Soret spectra of carboxycytochrome P-450

Cited by 187 publications

References 1 publication

Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam

Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam

Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies

Carbon monoxide binding to pentacoordinate mercaptide-heme complexes: kinetic study on models for cytochrome P-450.

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Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam