Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate

Groves, John T.; McClusky, Gary A.; White, Ronald E.; Coon, Minor J.

doi:10.1016/0006-291x(78)91643-1

Cited by 537 publications

(269 citation statements)

References 17 publications

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“…An observed 2 H KIE for Ole-I decay at this H 2 O 2 concentration, which represents a lower limit for the unmasked value, is provided by a ratio of the fast RRTs, [ H k app / D k app = RRT 1 (H 39 -EA)/RRT 1 (D 39 -EA)], giving a KIE ≥ 8.1 ± 1.1. The large KIE, which has not been previously measured for a P450-I generated with a bound substrate, is in accord with unmasked KIEs for steady-state P450 hydroxylations (12), rapid mixing studies of P450-I (10), and similar thiolateligated heme iron−oxo species (41), and theoretical considerations (43). This confirms that the distinctive C−Cα cleavage catalyzed by OleT most likely commences in a very similar way to P450 monooxygenations.…”

Section: Significancesupporting

confidence: 78%

“…The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11,12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs.…”

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confidence: 99%

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Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Grant

Mitchell

Makris

2016

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

102

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OleT is a cytochrome P450 that catalyzes the hydrogen peroxidedependent metabolism of C n chain-length fatty acids to synthesize C n-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe 4+ −oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe 4+ −OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.C ytochrome P450 (CYP) enzymes catalyze an extraordinary breadth of physiologically important oxidations for xenobiotic detoxification and specialized biosynthetic pathways (1, 2). The metabolic diversity of CYP enzymes originates from a sophisticated interplay of substrate molecular recognition with precise tuning of metal oxygen species formed at the enzyme active site. CYPs use a thiolate-ligated heme iron cofactor to activate molecular oxygen and produce short-lived ferric superoxo (3-5), ferric (hydro)peroxo (6-8), and ferryl (9, 10) intermediates. Coordinated efforts over several decades have resulted in isolation of each intermediate, including recent characterization of the principal oxidant thought to be responsible for the vast majority of P450 oxidations, the Fe 4+ −oxo pi−cation radical species commonly referred to as compound I (or P450-I) (10).The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11, 12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs. 13-17). Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.P450-I has also been link...

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Section: Significancesupporting

confidence: 78%

mentioning

confidence: 99%

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Grant

Mitchell

Makris

2016

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

102

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“…Furthermore, steric hindrance in a substrate molecule by itself against attacking active oxygen species in cyt P-450 would similarly affect the relative ease of metabolic hydroxylation. The superiority of the fR index in most cases studied here suggests that metabolic hydroxylation catalyzed by cyt P-450 proceeds via the triplet (radical) mechanism, which is in agreement with studies on the oxidation of norbornane by a reconstituted liver cyt P-450 system reported by Groves et al 10) 2. Metabolic Hydroxylation at the Aromatic C-H Bonds Since metabolic hydroxylation is considered to occur at the C-H moiety, carbon atoms not linked to hydrogen were excluded from the estimates of site specificity.…”

Section: Metabolic Hydroxylation At Aliphatic C-hsupporting

confidence: 92%

Theoretical Estimation of Site Specificity in the Metabolic Hydroxylation of Synthetic Pyrethroids

Katagi

1990

J. Pestic. Sci.

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Site specificity in the metabolic hydroxylation of pyrethroid insecticides catalyzed by monooxygenases was theoretically estimated by AM1 calculations. The radical reaction index fR of hydrogen, derived from frontier orbital theory, could mostly explain site specificity in the metabolic hydroxylation of aliphatic moieties, while the Mulliken overlap population of an individual C-H bond afforded erroneous results.Site specificity in the metabolic hydroxylation of aromatic moieties was successfully related to the electrophilic reaction index fE of carbon.

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“…The reactive intermediate is formed by the reduction of oxygen bound to the heme iron followed by protonation and heterolytic scission of the dioxygen bond with elimination of the water molecule and formation of a highly electrophilic oxygen atom bound to the hypervalent heme iron. Oxygenation of aliphatic C-H bonds is thought to proceed by abstraction of a hydrogen by the iron oxo intermediate followed by recombination of the resulting substrate radical with the transiently iron-bound hydroxyl radical (14). As the strength of primary C-H bonds is greater than that of secondary C-H bonds, oxygen addition at the -1 or -2 positions of fatty acids is typically seen for less specialized enzymes (15)(16)(17).…”

mentioning

confidence: 99%

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

Hsu

Baer

Rettie

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeP450 family 4 fatty acid -hydroxylases preferentially oxygenate primary C-H bonds over adjacent, energetically favored secondary C-H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for -hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C-H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for -hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for -hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane -hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for -hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for -hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C-H bonds.Cytochrome P450 family 4 fatty acid -hydroxylases are heme-containing monooxygenases that provide pathways for the reduction of potentially toxic non-esterified fatty acids and for the elimination of excess nutritive and non-nutritive lipids by catalyzing the addition of oxygen to a C-H bond of the terminal -carbon of fatty acids (1, 2). The resulting -alcohols are generally oxidized further to produce dicarboxylic acids, but they also may be transformed to glucuronides or esterified to glycerolipids. Urinary dicarboxylic fatty acids are elevated in humans by fasting or in uncontrolled diabetes, which are conditions where adipocyte lipolysis increases the availability of non-esterified fatty acids to fuel metabolism (3). It is estimated that roughly 15% of fatty acids undergo -hydroxylation during peak periods of fatty acid catabolism (4), whereas this fraction is much smaller under conditions where concentrations of nonesterified fatty acids are low (5). Collectively, these enzymes provide pathways for the metabolic clearance of branched chain fatty acids, eicosanoids, and xenobiotic substrates such as dietary phytanic acid, drugs, toxins, and vitamins E and K (1, 2, 6).Similar to other P450 2 gene families encoding multipurpose enzymes for the elimination of xenobiotic compounds, family 4 exhibits genetic diversi...

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Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate

Cited by 537 publications

References 17 publications

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Theoretical Estimation of Site Specificity in the Metabolic Hydroxylation of Synthetic Pyrethroids

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

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