Cytochrome P450Dependent Steroid Hydroxylase Systems

Cytochromes P450 got their name from both their hemoprotein character and from their unusual spectral properties, displaying a typical absorption maximum of the reduced CO-bound complex at 450 nm: cytochrome stands for a hemopro-

tein, P for pigment and 450 reflects the absorption peak of the CO complex at 450 nm. The ability of reduced P450 to produce an absorption peak at 450 nm upon CO binding is still used for the estimation of the P450 content [3]. These proteins are encoded by a superfamily of genes, convert a broad variety of substrates and catalyze a variety of interesting chemical reactions. This enzyme family is involved in the biotransformation of drugs, the bioconversion of xeno-biotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins, bile acids, the conversion of alkanes, terpenes, and aromatic compounds as well as the degradation of herbicides and insecticides. There is also a wide range of reactions catalyzed by cytochromes P450, such as carbon hydroxylation, hetero-atom oxygenation, dealkylation, epoxidation, aromatic hydroxylation, reduction, dehalogenation (reviewed in [4-6]).

To date, more than 6000 different P450 genes have been cloned up to date (for details see: http://drnelson.utmem.edu/CytochromeP450.html). Members of the same gene family are defined as usually having >40% sequence identity with a P450 protein from any other family. Mammalian sequences within the same subfamily are always >55% identical. The numbers of individual P450 enzymes in different species differ significantly, the highest numbers observed so far being displayed in plants.

Cytochromes P450 are external monooxygenases (mixed function oxidases) since they catalyze the incorporation of a single atom of molecular oxygen into a substrate with the concomitant reduction of the other atom to water. The monooxygenases are divided into two classes, the internal and the external monooxygenases. Internal monooxygenases extract two reducing equivalents from the substrate to reduce one atom of dioxygen to water, whereas external monooxygen-ases utilize an external reductant (see [7]). Cytochrome P450 systems catalyze the following reaction:

Details of the structure and chemistry of cytochromes P450 have recently been summarized in an excellent review [8] and for this reason will not be discussed here. It should be mentioned, however, that cytochromes P450 do not only catalyze monooxygenations, but also oxidase and peroxidase reactions. Variations of this scheme for the reaction mechanism of P450, however, occur with different P450 systems such as thromboxane and prostacyclin synthase, nitric oxide reductase (CYP55A1), and others. These different reaction types are, in addition to the main reaction cycle, the basis for the versatility of cytochromes P450.

As mentioned above, cytochromes P450 are external monooxygenases, which implies that they need an external electron donor to transfer the electrons necessary for oxygen activation and the subsequent substrate hydroxylation. Two main classes of cytochromes P450 can be defined with respect to their electron supporting system, although other subclasses also exist [9]: the mitochondrial/bacte-rial type and the microsomal type (Fig. 6.2). Microsomal cytochromes P450 are

Cytochrome P450 Cycle
Fig. 6.2 Schematic organization of different cytochrome P450 systems. (a) bacterial system, (b) mitochondrial system. (c) microsomal system, (d) self-sufficient CYP102 (P450BM-3). Figure slightly modified from [21].

membrane bound and accept electrons from a microsomal NADPH-cytochrome P450 reductase, containing FAD and FMN. All drug and xenobiotica metabolizing cytochromes P450 isolated so far and, in addition, CYP102 (P450BM-3) isolated from Bacillus megaterium, have been shown to belong to this class. CYP102 consists of a polypeptide chain with two different domains, one comprising the he-moprotein and the other containing an FAD- and FMN-dependent reductase.

Most of the other bacterial cytochromes P450 belong to the second class. They are soluble and obtain the electrons necessary for the reaction mechanism from an NADH-dependent FAD-containing reductase via an iron-sulfur protein of the [2Fe-2S] type. Mitochondrial cytochromes P450 involved in the side-chain cleavage of cholesterol, the 1ip-hydroxylation of 11-deoxycortisol, the production of aldosterone, and vitamin D biosynthesis also belong to the latter class. These cytochromes P450 are localized in the inner mitochondrial membrane, whereas the [2Fe-2S] protein called adrenodoxin (Adx) in adrenal steroid hydroxylase systems is a soluble protein of the matrix. The FAD-containing reductase adrenodoxin reductase (AdR) is associated with the inner mitochondrial membrane.

Interaction of the cytochromes P450 with their corresponding electron donors is a necessary prerequisite of the catalytic cycle. Its specificity guarantees a sufficient reaction rate of catalysis and likewise a discrimination between different potential donors and acceptors of electrons to protect the system from shunt reactions. Since in liver microsomes many different isoenzymes have to interact with only one type of reductase it has to be expected that the binding site for reductase is very similar or identical on various cytochromes P450. Salt bridges are responsible for the recognition of the reductase by the P450 and the correct orientation of both proteins to each other (for a review, see [10]). In addition to microsomal reductase, some microsomal cytochromes P450 are able to accept the second electron from cytochrome b5. Cytochrome b5 has also been shown to exert a differential stimulatory action, dependent upon both the form of cytochrome P450 and the reaction substrate [11, 12] (reviewed in [13, 14]).

In mitochondrial steroid hydroxylases and in the camphor hydroxylating bacterial P450 (CYP101) system a charge-pair interaction mechanism has been demonstrated by chemical modification, site-directed mutagenesis studies, and structural data of electron transfer complexes (for review, see [15]). In addition, the C-terminal peptide of adrenodoxin [16], the residue Tyr82 [17], and the loop covering the iron-sulfur cluster [18] were shown to be of pivotal importance for redox partner interaction. Like microsomal reductase, the mitochondrial ferredoxin has also to deliver electrons to different cytochromes P450. From the available data, a shuttle model is favored, where the oxidized ferredoxin interacts first with the ferre-doxin reductase to undergo reduction, with the formation of a Fe3+/Fe2+ iron-sulfur cluster [15]. It dissociates from the reductase and then interacts with the respective cytochrome P450, where it delivers this electron before returning to the reductase and transfers the second electron to the P450. The mechanism of electron transfer between the components of the different cytochrome P450 systems, one of the fundamental problems in life sciences, is not yet well understood. Although it could be conclusively shown that posttranslational modifications can regulate these electron transfer reactions at least in some cases [19], we are only at the very beginning of this field of research as well. Since electron transfer to the P450 in some cases seems to be low and rate-limiting in P450 catalysis [20], engineering of this step could potentially, however, lead to significantly improved biocatalysts. The current status of the biotechnological use as well as the possibilities of genetic engineering of cytochromes P450 were recently reviewed [21].

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