Magnetic circular dichroism studies. 44. Chloroperoxidase. Evidence for P-450 type heme environment from magnetic circular dichroism spectroscopy

Dawson, John H.; Trudell, James R.; Barth, Guenther; Linder, Robert E.; Bunnenberg, E.; Djerassi, Carl; Chiang, R.; Hager, Lowell P.

doi:10.1021/ja00428a055

Cited by 88 publications

(34 citation statements)

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“…CPO contains the heme iron prosthetic group found in most other peroxidases, but in contrast to other peroxidases, CPO does not have a proximal histidine heme iron ligand and instead shares with the P450 cytochromes the property of having a proximal heme iron thiolate ligand (18)(19)(20)(21)(22)(23)(24)(25). CPO also is unique in the hemoprotein family by virtue of having a glutamic acid residue distal to the heme-iron (25).…”

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confidence: 99%

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Mroczko

Manoj

et al. 1999

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

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Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

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confidence: 99%

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Mroczko

Manoj

et al. 1999

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

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“…As a result of the possession of a common proximal ligand, CPO and P450 share a range of spectral properties both with each other and with synthetic iron(III) porphyrins (hemins) containing a thiolate ligand (21), including optical (22,23), magnetic circular dichroism (24)(25)(26), electron paramagnetic resonance (EPR) (27)(28)(29), ENDOR (30), and Mössbauer (31,32) properties. Interestingly, even though CPO and P450 possess a similar active-site structure and similar activities, the catalytic cycles of the two enzymes are different.…”

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confidence: 99%

Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene

Debrunner

Dexter

Schulz

et al. 1996

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

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We have used Mössbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-Nalkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme (''green CPO'') displayed a nearly pure low-spin ferric EPR signal with g ‫؍‬ 1.94, 2.15, 2.31. The Mössbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three 2 T 2 levels of the (3d) 5 configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the 6 A 1 states follow the 2 T 2 ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green CPO to 22؇C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S ‫؍‬ 1 ⁄2 ferric species. One of these species displayed g ‫؍‬ 1.82, 2.25, 2.60 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g ‫؍‬ 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this ''quasi-native CPO,'' which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.

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“…Recent studies show that CPO is able to catalyze P450-like monooxygenase reactions (Fig 1.7) (Sundaramoorthy 1995). Finally, CPO has been shown to catalyze the dehalogenation of selected halogenated aromatic compounds (Dawson 1976).…”

Section: Cpo-catalyzed Reactionsmentioning

confidence: 99%

“…Therefore, carbon monoxide is an efficient substrate to identify the proximal ligands of hemoproteins (Sono 1996;Ortiz de Montellano 1998;Omura 2005). arbon e has been confirmed to be a thiolate anion (Dawson 1976), but the P450-like "heme-binding region" has not been identified in CPO's primary sequence (Fang 1986). …”

Section: Imidazole-and Thiolate-ligated Hemoproteinsmentioning

confidence: 99%

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Jiang¹

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Magnetic circular dichroism studies. 44. Chloroperoxidase. Evidence for P-450 type heme environment from magnetic circular dichroism spectroscopy

Cited by 88 publications

References 1 publication

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

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