Cytochromes P450: a success story

Abstract

Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.

Figure 1

Primary structures of P450 proteins. (a) Typical features of an ER-bound P450 protein (class II enzyme). The function of the different domains and regions indicated by colors are described in the text. (b) Variants of this canonical structure most commonly found: 1, soluble class I; 2, mitochondrial class I; 3, membrane-bound or plastidial class III. The three-dimensional folding of these structures can be viewed at [17,18]. A good (model) picture of membrane-bound P450 can be seen at [36].

Figure 2

Secondary and tertiary structures of P450 proteins. (a) Topology diagram showing the secondary structure and arrangement of the secondary structural elements of a typical P450 protein (CYP102) [14]. Blue boxes, α helices; groups of cream arrows outlined with dotted lines, β sheets; lines, coils and loops. The sizes of the elements are not in proportion to their length in the primary sequence. There are usually around four β sheets and 13 α helices defining one domain that is predominantly β sheets and one that is predominantly α helices. The first domain is often associated with substrate recognition and the access channel, the second with the catalytic center. Adapted from [14]. (b) A ribbon representation of the distal face of the folded CYP2C5 protein showing its putative association with the ER membrane (purple) [16]. Helices and sheets are labeled as in (a). Heme is in orange, the substrate in yellow. The α domain is on top left, the β domain more closely associated with the membrane at bottom right. Epitopes not accessible for antibody binding when the protein is associated with the ER are shown in red (numbers give their position in the primary sequence). The transmembrane amino-terminal segment, removed for crystallization, and an additional II residues that are disordered in the crystal structure, are not shown. Note the I helix above the heme, close to the substrate-binding site. The heme-binding loop is visible behind the heme protoporphyrin. The conserved Gln-X-X-Arg structure in the K helix is also at the back and so is not readily visible. The proximal (back) face of the protein is involved in redox partner recognition and electron transfer to the active site; protons flow into the active site from the distal face (front). The substrate access channel is usually assumed to be located in close contact of the membrane between the F-G loop, the A helix and β strands 1-1 and 1-2. More pictures showing other aspects of the structure, including reductase and substrate-binding, can be viewed at [17,18]. Another picture (a model) of membrane-bound P450 including the transmembrane domain can be seen at [36]. Reproduced with permission from [37].

Figure 3

Catalytic mechanism of P450 enzymes. P450s are usually mono-oxygenases, catalyzing the insertion of one of the atoms of molecular oxygen into a substrate, the second atom of oxygen being reduced to water. The most frequently catalyzed reaction is hydroxylation (O insertion) using the very reactive and electrophilic iron-oxo intermediate (species [C], bottom row). The hydroperoxo form of the enzyme (species [B]^-) is also an electrophilic oxidant catalyzing OH⁺ insertion. Nucleophilic attack can be catalyzed by species [A]^2- and [B]^- ; reduction, isomerization or dehydration are catalyzed by the oxygen-free forms of the enzyme. This, together with the variety of the apoproteins and intrinsic reactivity of all their substrates explains the extraordinary diversity of reactions catalyzed by P450 enzymes.