ion phase that leads to an alkyl radical coordinated to the iron-hydroxo complex by a weak OH---C hydrogen bond, labeled as CI; (ii) an alkyl (or OH) rotation phase whereby the alkyl group achieves a favorable orientation for rebound; and (iii) a rebound phase that leads to C-O bond making and the ferric-alcohol complexes, 4,2P. The two profiles remain close in energy throughout the first two phases and then bifurcate. Whereas the HS state exhibits a significant barrier and a genuine TS for rebound, in the LS state, once the right orientation of the alkyl group is achieved, the LS rebound proceeds in a virtually barrier-free fashion to the alcohol. As such, alkane hydroxylation proceeds by TSR,70-72,120 in which the HS mechanism is truly stepwise with a finite lifetime for the radical intermediate, whereas the LS mechanism is effectively concerted with an ultrashort lifetime for the radical intermediate. Subsequent studies of ethane and camphor hydroxylation by the Yoshizawa group117,181-183 arrived at basically the same conclusion, that the mechanism is typified by TSR. The differences between the results of Shaik et al.130,173,177-180 and Yoshizawa et al.117,181-183 were rationalized recently71,72 and shown to arise owing to technical problems and the choice of the mercaptide ligand,117,181-183 which is a powerful electron donor and is too far from the representation of cysteine in the protein environment. The most recent study of camphor hydroxylation, which was done at a higher quality,117 converged to the picture reported by Shaik et al.130,173,177-180 and shows a stepwise HS process with a barrier of more than 3 kcal/mol for C-O bond formation by rebound of the camphoryl radical vis-à-vis an effectively concerted LS process for which this barrier is 0.7 kcal mol-1 and is the rotational barrier for reaching the rebound position. By referring to Figure 21, it is possible to rationalize the clock data of Newcomb in a simple manner. The apparent lifetimes are based on the assumption that there is a single state that leads to the reaction, such that the radical lifetime can be quantitated from the rate constant of free radical rearrangement and the ratio of rearranged to unrearranged alcohol product. However, in TSR, the rearranged (R) product is formed only/mainly on the HS surface, while the unrearranged (U) product is formed mainly on Figure 20. Formal descriptions of iron(III)-peroxo, iron(III)-hydroperoxo, and iron(V)-oxo species with indication of the negative charges. The roles “electrophile” or “nucleophile” are assigned according to the charge type. Reprinted with permission from ref 7. Copyright 2000 Springer-Verlag Heidelberg. 3964 Chemical Reviews, 2004, Vol. 104, No. 9 Meunier et al.

Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes.

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure.

Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

http://science.sciencemag.org/content/sci/287/5458/1615.full.pdf

The Catalytic Pathway of Cytochrome P450Cam at Atomic Resolution

Theoretical perspective on the structure and mechanism of cytochrome P450 enzymes.

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

Activity of methionine synthetase was measured in liver biopsies of seven patients who had received 50% to 70% nitrous oxide supplemented by a combination of a narcotic and/or barbiturate with or without a volatile anesthetic, and from seven patients who were anesthetized without nitrous oxide (control group). Methionine synthetase activity (+/- SE) averaged 219 +/- 28 nmol of methionine per hour per gran of liver in patients given nitrous oxide, and 414 +/- 29 in control patients. Inactivation of methionine synthetase progressively increased as the product of the concentration of nitrous oxide and the exposure time increased. These results in humans are similar to those in animals and suggest that inactivation of methionine synthetase may play a role in the development of the pathologic effects seen in patients and medical personnel after exposure to nitrous oxide.

Nitrous Oxide Inactivates Methionine Synthetase in Human Liver

We examined the metabolism of desflurane in 13 healthy volunteers given 7.35 +/- 0.81 MAC-hours (mean +/- SD) of desflurane and 26 surgical patients given 3.08 +/- 1.84 MAC-hours (mean +/- SD). Markers of desflurane metabolism included fluoride ion measured via an ion-specific electrode, nonvolatile organic fluoride measured after sodium fusion of urine samples, and trifluoroacetic acid determined by a gas chromatographic-mass spectrometric method. In both volunteer and patient groups, postanesthesia serum fluoride ion concentrations did not differ from background fluoride ion concentrations. Similarly, postanesthesia urinary excretion of fluoride ion and organic fluoride in volunteers was comparable to preanesthesia excretion rates. However, small but significant levels of trifluoroacetic acid were found in both serum and urine from volunteers after exposure to desflurane. A peak serum concentration of 0.38 +/- 0.17 mumol/L of trifluoroacetic acid and a peak urinary excretion rate of 0.169 +/- 0.107 mumol/h were detected in volunteers at 24 h after desflurane exposure. Although these increases in trifluoroacetic acid after exposure to desflurane were statistically significant, they are approximately 10-fold less than levels seen after exposure to isoflurane. Thus, desflurane strongly resists biodegradation, but a small amount is metabolized in humans.

Fluoride metabolites after prolonged exposure of volunteers and patients to desflurane.

The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

The optimized geometries of the stable ferrous dioxygen and transient reduced ferrous dioxygen forms of a methylmercaptate porphine model of the cytochrome P450 heme system were calculated using nonlocal density functional theoretical (DFT) methods. The optimized geometry of the ferrous dioxygen form is in good agreement with the structure of a model compound of this species and the calculated diamagnetic singlet ground state is consistent with the reported lack of ESR spectrum of this species. The calculated ground state of the reduced ferrous dioxygen species is a low-spin (doublet) state, in agreement with the reported ESR signature of this species in P450cam. The dioxygen ligand in the reduced form is shown to preferentially bind to the heme iron in an asymmetric “end-on” geometry. The most pronounced structural effects of the reduction of the ferrous dioxygen species are the elongation of the Fe−O and Fe−S bonds. This bond lengthening is due to the addition of an electron into a molecular orbital of ...

Lucy Waskell

Papers

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

Nitrous Oxide Inactivates Methionine Synthetase in Human Liver

Fluoride metabolites after prolonged exposure of volunteers and patients to desflurane.

The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

Structure and Spectra of Ferrous Dioxygen and Reduced Ferrous Dioxygen Model Cytochrome P450