Heme-Containing Oxygenases

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.

Two decades have passed since the discovery in liver microsomes of a haemprotein that forms a reduced-CO complex with the absorptive maximum of the Soret at 450 nm (Klingenberg, 1958; Garfinkel, 1958) and the identification of this protein as a new cytochrome: pigment cytochrome, P-450 (Omura and Sato, 1962, 1964a). In the intervening years, the study of cytochrome P-450 dependent monoxygenases has expanded exponentially. From the first crude attempts to solubilise a P-450 (Omura and Sato, 1963, 1964b) to the determination of the primary, secondary, and tertiary structure of cytochrome P-450cam by amino acid sequencing (Haniu et al., 1982a,b) and x-ray crystallography (Poulos et al., 1984) our understanding of this unique family of proteins has been advancing on all fronts. Since, perhaps, the greatest understanding of the structure and mechanism of P-450s has come from concentrated study of P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, this review will concentrate on findings with P-450cam; attention will be drawn to parallel and contrasting examples from other P-450s as appropriate.

Structure and Chemistry of Cytochrome P450

The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, and many important papers on the roles of P450s in chemical toxicology have appeared in Chemical Research in Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established, in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structures of several of the major human P450s are now in hand. The animal P450s are still important in the context of metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Unresolved problems include the characterization of the minor “orphan” P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction of metabolism, the overall contribution of bioactivation to drug idiosyncratic probl...

Cytochrome P450 and Chemical Toxicology

High-resolution crystal structure of cytochrome P450cam.

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

http://deepblue.lib.umich.edu/bitstream/handle/2027.42/21710/0000102.pdf?sequence=1

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

https://portlandpress.com/biochemsoctrans/article-pdf/3/6/813/520300/bst0030813.pdf

Purified Liver Microsomal Cytochrome P -450: Catalytic Mechanism and Characterization of Multiple Forms

Evidence has been obtained from enzyme fractionation that liver microsomal cytochrome P–450 (P–450 LM ) exists in multiple forms. Two of these have been isolated in a homogeneous state. They are designated according to their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P–450 LM 2 and P–450 LM 4 . P–450 LM 2 , which was isolated from phenobarbital–induced rabbits, has a subunit molecular weight of 48,700, and P–450 LM 4 from β–naphthoflavone–induced rabbits has a subunit molecular weight of 55,300. These proteins also differ in their optical and electron paramagnetic resonance spectra, catalytic activities, and other properties. Studies on the mechanism of action of P–450 LM 2 have shown that it produces hydrogen peroxide during substrate hydroxylation in the presence of molecular oxygen in a reconstituted enzyme system containing NADPH, NADPH–cytochrome P–450 reductase, and phosphatidylcholine. Peroxides also serve as oxygen donors for substrate hydroxylation, catalyzed by P–450 LM 2 in the absence of molecular oxygen, NADPH, and the reductase. P–450 LM 2 is a two–electron acceptor under anaerobic conditions; the reduced protein reacts with O 2 to form two different oxyferro complexes, as shown by stopped flow methods. The rate of decomposition of the second complex appears to be altered by the presence of cytochrome b 5 or NADPH–cytochrome P–450 reductase. A scheme is presented to account for these and other related findings on the mechanism of P–450 LM –catalyzed hydroxylation reactions.

Purification of membrane–bound oxygenases: isolation of two electrophoretically homogeneous forms of liver microsomal cytochrome p-450

The purpose of this paper is to summarize our knowledge of the properties of purified liver microsomal cytochrome P-450, including evidence for multiple forms, and to present data from recent experiments bearing on the mechanism of action of this cytochrome. For the sake of brevity we will confine our discussion to studies in this laboratory on cytochrome P-450 solubilized and purified from rabbit liver microsomes.

Ronald E. White

Papers

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

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

Purified Liver Microsomal Cytochrome P -450: Catalytic Mechanism and Characterization of Multiple Forms

Purification of membrane–bound oxygenases: isolation of two electrophoretically homogeneous forms of liver microsomal cytochrome p-450

Highly Purified Cytochrome P-450 from Liver Microsomal Membranes: Recent Studies on the Mechanism of Catalysis