4-Cyanopyridine, a Versatile Spectroscopic Probe for Cytochrome P450 BM3

Ost, Tobias W B; Clark, Jonathan P.; Anderson, J. L. Ross; Yellowlees, Lesley J.; Daff, Simon; Chapman, Stephen K

doi:10.1074/jbc.m408601200

Cited by 18 publications

(25 citation statements)

References 30 publications

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“…It should be noted that addition of the C 16 -fatty acid to a buffered assay system containing hemoprotein incorporated into a pyrolytic graphite electrode failed to alter the cyclic voltammetric peaks [78]. Moreover, the weakly bound C 12 -substrate, laurate, again generating a mixture of spin forms, causes a change in E 0' much smaller than that ascribed to the action of palmitate [67,79]. Similar disparates were found with some CYP102A1 mutants.…”

Section: The Cyp102 and Cyp106 Enzymesmentioning

confidence: 48%

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading toxic pollutants. Finally, availability of P450 nanobiochips for high-throughput screening of substrate libraries might expedite drug development.

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Section: The Cyp102 and Cyp106 Enzymesmentioning

confidence: 48%

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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“…An interesting feature of the P450 101A1 and 102A1 systems is that the binding of substrate has been demonstrated to lower the E m,7 (78,104,105). As discussed (see above), this change indicates that the substrate binds more tightly to the ferrous enzyme (74,79 complex (Fig. 7C).…”

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confidence: 90%

“…2, 6). Very recently Ost et al (79) reported that the ligand 4-cyanopyridine is bound 3 orders of magnitude more tightly to ferrous than ferric P450 102A1, and accordingly, the presence of this ligand lowers the E m,7 .…”

mentioning

confidence: 99%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

115

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Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

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“…Anaerobic potentiometric titrations were carried out as described (34) at 25°C by using a modified quartz EPR OTTLE cell. Titrations were performed in both the absence and presence of L-Trp (15 mM), and the heme reduction potentials were determined by fitting the data to the Nernst equation for a single-electron process by using Origin software (MicroCal, Northampton, MA).…”

Section: Methodsmentioning

confidence: 99%

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

Forouhar

Anderson

Mowat

et al. 2007

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

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169

284

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Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.cancer ͉ heme enzymes ͉ immunomodulation ͉ indoleamine 2,3-dioxygenase T ryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring, the first and rate-limiting step in L-Trp catabolism through the kynurenine pathway (1-3). In addition, IDO has been implicated in a diverse range of physiological and pathological conditions, including suppression of T cell proliferation, maternal tolerance to allogenic fetus, and immune escape of cancers (4-8), and is an attractive target for drug discovery against cancer and autoimmune and other diseases (2, 9-12).Despite catalyzing identical biochemical reactions (Fig. 1a), the sequence similarity between TDO and IDO is extremely low. An alignment of their sequences is only possible based on their structures, which suggests a sequence identity of 10% between them (Fig. 1b). In comparison, Xanthomonas campestris TDO shares 34% sequence identity with human TDO (Fig. 1b), demonstrating the remarkable evolutionary conservation of this enzyme. TDO is a homotetrameric enzyme and is highly specific for L-Trp and related derivatives such as 6-fluoro-Trp as the substrate. In comparison, IDO is monomeric, and shows activity toward a larger collection of substrates, including L-Trp, Dtryptophan (D-Trp), serotonin, and tryptamine (3), although the K m for D-Trp is Ϸ100-fold higher than that for L-Trp (13). The structure of human IDO in the catalytically inactive, ferric [Fe(III)]-heme state in complex with the 4-phenylimidazole inhibitor has recently been reported (14). Although this structure gave information about important active site residues, the inhibitor is coordinat...

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4-Cyanopyridine, a Versatile Spectroscopic Probe for Cytochrome P450 BM3

Cited by 18 publications

References 30 publications

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

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