Calculated and experimental absolute stereochemistry of the styrene and .beta.-methylstyrene epoxides formed by cytochrome P 450cam

Fruetel, Julia A.; Collins, Jack; Camper, Debra L.; Loew, Gilda H.; Montellano, Paul R. Ortiz de

doi:10.1021/ja00044a006

Cited by 112 publications

(92 citation statements)

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“…Putidaredoxin and putidaredoxin reductase were expressed and purified following standard literature methods [13,14]. The concentration of hydrogen peroxide in camphor oxidation incubations was determined by literature methods [15,16].…”

Section: Generalmentioning

confidence: 99%

“…97%) coupling efficiencies whether or not potassium was present (data not shown). The amount of hydrogen peroxide generated by any uncoupling of the catalytic cycle for camphor oxidation was determined using literature methods [15,16]. Camphor oxidation catalysed by wild-type P450 cam and the mutants all showed , 3% uncoupling via peroxide generation, whether or not potassium was present (data not shown).…”

Section: Camphor Oxidation Activitymentioning

confidence: 99%

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Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam

Westlake¹,

Harford-Cross²,

Donovan³

et al. 1999

European Journal of Biochemistry

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Cytochrome P450 cam (CYP101) from Pseudomonas putida is unusual among P450 enzymes in that it exhibits co-operative binding between the substrate camphor and a potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a surface residue which forms part of the cation-binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co-operativity of potassium and camphor binding by P450 cam , and also to influence the catalytic activity. In particular, replacement of Glu84 by positively charged residues such as lysine results in increased high-spin haem fractions and camphor turnover activities in the absence of potassium, along with decreased camphor dissociation constants. However, in the presence of potassium the camphor dissociation constants of these mutants are significantly increased compared with the wild-type, although the camphor turnover activities remain marginally higher. In contrast, substitution by aspartate results in tighter binding of both potassium and camphor, but has little effect on the enzymatic activity. In all cases the reaction remains essentially 100% coupled and gives 5-exo-hydroxycamphor as the only product. These results suggest that an anionic side chain at the 84 position is crucial for the co-operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450 cam is to promote camphor binding even at the expense of turnover rate, thus allowing the organism to utilize low environmental concentrations of this substrate for growth.Keywords: cations; co-operativity; cytochrome P450 cam ; electron transfer; potassium.It has long been recognized that cations, and potassium in particular, play an important role in the chemistry of cytochrome P450 cam (CYP101) from Pseudomonas putida [1,2]. Potassium stabilizes P450 cam against conversion to the P420 form [3,4], promotes camphor binding and, perhaps most importantly, is required to drive the haem in the camphor-bound protein to 100% high-spin at physiological pH [5]. A linear relationship has been observed between the free energies of potassium and camphor binding, thus establishing the cooperativity of the two processes [6]. As the spin-state equilibrium affects both the haem redox potential and the rate of the first electron transfer [1,2], potassium binding optimizes the enzyme for camphor oxidation.The high-resolution crystal structure of P450 cam revealed a potential potassium-binding site and a possible mechanism of action [7]. An ordered solvent molecule,`Water-515', was found in an almost perfect octahedral environment consisting of the backbone carbonyls of E84, G93, E94, and Y96, and two ordered solvent molecules (Water-517 and Water-584). All six O´´´O distances were less than 3 A Ê , and one of the ordered water molecules was hydrogen-bonded to the carboxylate side chain of E84 (Fig. 1). This octahedral environment strongly suggested that Water-515 was actually a protein-bound cation. The size of the cation binding si...

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Section: Generalmentioning

confidence: 99%

Section: Camphor Oxidation Activitymentioning

confidence: 99%

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam

Westlake¹,

Harford-Cross²,

Donovan³

et al. 1999

European Journal of Biochemistry

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“…[8][9][10][11][12][13][14][15][16][17] Styrene is epoxidized by synthetic oxo-metal porphyrin catalysts as well as by wild-type P450s. [18,19] In particular, Vaz et al [19] measured rate constants for styrene epoxidation in several P450 isozymes and mutants, and estimated free energy barriers of 24-25 kcal mol…”

Section: Introductionmentioning

confidence: 99%

“…d) The high-spin radical intermediate, 4 2 rad -III, has a substantial ring closure barrier and may survive long enough time to lead to suicidal, phenacetaldehyde and 2-hydroxostyrene side products. e) The phenacetaldehyde and 2-hydroxostyrene products both originate from crossover from the side the epoxide also phenacetaldehyde [10,17,18] and the N-alkylated porphyrin complex. The latter species, the so-called suicidal complex, destructs the catalyst/enzyme by forming dead-end products that do not allow regeneration of the active species.…”

mentioning

confidence: 99%

Multistate Reactivity in Styrene Epoxidation by Compound I of Cytochrome P450: Mechanisms of Products and Side Products Formation

Kumar

Visser

Shaik

2005

Chemistry A European J

108

102

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Density functional theoretical calculations are used to elucidate the epoxidation mechanism of styrene with a cytochrome P450 model Compound I, and the formation of side products. The reaction features multistate reactivity (MSR) with different spin states (doublet and quartet) and different electromeric situations having carbon radicals and cations, as well as iron(III) and iron(IV) oxidation states. The mechanisms involve state-specific product formation, as follows: a) The low-spin pathways lead to epoxide formation in effectively concerted mechanisms. b) The high-spin pathways have finite barriers for ring-closure and may have a sufficiently long lifetime to undergo rearrangement and lead to side products. c) The high-spin radical intermediate, (4)2(rad)-IV, has a ring closure barrier as small as the C--C rotation barrier. This intermediate will therefore lose stereochemistry and lead to a mixture of cis and trans epoxides. The barriers for the production of aldehyde and suicidal complexes are too high for this intermediate. d) The high-spin radical intermediate, (4)2(rad)-III, has a substantial ring closure barrier and may survive long enough time to lead to suicidal, phenacetaldehyde and 2-hydroxostyrene side products. e) The phenacetaldehyde and 2-hydroxostyrene products both originate from crossover from the (4)2(rad)-III radical intermediate to the cationic state, (4)2(cat,z(2) ). The process involves an N-protonated porphyrin intermediate that re-shuttles the proton back to the substrate to form either phenacetaldehyde or 2-hydroxostyrene products. This resembles the internally mediated NIH-shift observed during benzene hydroxylation.

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“…The enzyme catalyzes camphor hydroxylation at the 5-exo position and requires NADH as well as two protein cofactors. Stereospecific hydroxylation of other substrates has been investigated (4,5). As shown in Fig.…”

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confidence: 99%

A direct electrode-driven P450 cycle for biocatalysis

Reipa

Mayhew²,

Vilker³

1997

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

106

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The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor͞nmol of CYP101 per min.

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Calculated and experimental absolute stereochemistry of the styrene and .beta.-methylstyrene epoxides formed by cytochrome P 450cam

Cited by 112 publications

References 1 publication

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam

Multistate Reactivity in Styrene Epoxidation by Compound I of Cytochrome P450: Mechanisms of Products and Side Products Formation

A direct electrode-driven P450 cycle for biocatalysis

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Calculated and experimental absolute stereochemistry of the styrene and .beta.-methylstyrene epoxides formed by cytochrome P 450cam

Cited by 112 publications

References 1 publication

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450cam

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450cam

Multistate Reactivity in Styrene Epoxidation by Compound I of Cytochrome P450: Mechanisms of Products and Side Products Formation

A direct electrode-driven P450 cycle for biocatalysis

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Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam

Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450_cam