4 Electron Transfer Partners of Cytochrome P450 Mark J.l. Paine, Nigel S. Scrutton, Andrew W. Munro, Aldo Gutierrez, Gordon C.K. Roberts, and C. Roland Wolf 1. Introduction Although P450 redox partners are usually expressed independently, "self-sufficient" P450 monooxygenase systems have also evolved through Cytochromes P450 contain a heme center the fusion of P450 and CPR genes. These fusion where the activation of molecular oxygen occurs, molecules are found in bacteria and fungi, the best- resulting in the insertion of a single atom of known example being P450 BM3, a fatty acid oxygen into an organic substrate with the con­ (0-2 hydroxylase from Bacillus megaterium, which comitant reduction of the other atom to water. The comprises a soluble P450 with a fiised carboxyl- monooxygenation reaction requires a coupled and terminal CPR module (recently reviewed by stepwise supply of electrons, which are derived Munro^). BM3 has the highest catalytic activity from NAD(P)H and supplied via a redox partner. known for a P450 monooxygenase^ and was for P450s are generally divided into two major classes many years the only naturally occurring ftised sys­ (Class I and Class II) according to the different tem known until the identification of a eukaryotic types of electron transfer systems they use. P450s membrane-bound equivalent fatty acid hydroxy­ in the Class I family include bacterial and mito­ lase, CYP505A1, from the phytopathogenic fungus chondrial P450s, which use a two-component Fusarium oxysporurrP. A number of novel P450 sys­ shuttle system consisting of an iron-sulfur protein tems are starting to emerge from the large numbers (ferredoxin) and ferredoxin reductase (Figure 4.1). of genome sequencing projects now underway^. The Class II enzymes are the microsomal P450s, which receive electrons from a single mem­ In this chapter, we review the most recent brane-bound enzyme, NADPH cytochrome P450 advances being made in understanding the func­ reductase (CPR), which contains FAD and FMN tion of P450 redox partners and the electron trans­ cofactors (Figure 4.1). Cytochrome b^ may also fer process. Special attention is paid to CPR, couple with some members of the Class II P450s which occupies a particularly important position family, notably CYP3A4, to enhance the rate of because of its central involvement in human drug catalysis ^ metabolism. Mark J.l. Paine and C. Roland Wolf Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK. Nigel S. Scrutton Andrew W. Munro, Gordon C.K. Roberts and Aldo Gutierrez Department of Biochemistry, University of Leicester, Leicester, UK. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano Kluwer Academic / Plenum Publishers, New York, 2005. 115 116 Mark J.I. Paine et al. Class I: Iron-sulfur partners Adx/AdR Pdx/PdR NADPH NADP NAD(P)H NAD(P)* i FADp^\mQ) Mitochondrial membrane Class II: Diflavin reductase partners CPR CPR fusion NADPH NADP NADPH NADP 4 4 Novel Systems Fe2S2 fusion NAD* NADH Figure 4.1. Electron transfer partners of cytochrome P450. In Class I systems, electrons are shuttled from NAD(P)H through an FAD-containing ferredoxin reductase and an iron-sulfur containing ferredoxin to P450; in prokaryotes these are typified by putidoredoxin reductase (PdR) and putidoredoxin (Pdx), and in eukaryotes by mitochondrial membrane associated adrenodoxin reductase (AdR) and adrenodoxin (Adx). Class II systems are driven by electrons delivered from NADPH through diflavin (FMN- and FAD-containing) reductases. In eukaryotes these are bound to the endoplasmic reticulum, while fused systems such as P450 BM3 exist in bacteria and fungi. Novel systems now include P450RhF, which contains an FMN-containing reductase fused with a ferredoxin-like center and a P450. 2. NADPH-Cytochrome P450 apoptosis and cellular homeostasis^' ^, it is inter­ Reductase and the Diflavin esting that CPR was first isolated from yeast as an Reductase Family FMN containing NADPH-dependent cytochrome c reductase^. A mammalian equivalent was later 2.1. Background isolated from pig liver and reported to contain an FAD cofactor^. By 1962, the enzyme was shown In view of the key role that cytochrome c to be localized at the endoplasmic reticulum^, and has recently been found to play in regulating the flavin cofactors to be involved in the reduction Electron Transfer Partners of Cytochrome P450 117 of cytochrome c^^. The true physiological redox A useful feature of flavins is that their absorption partner was eventually discovered as P450 through spectra are altered by changes in their reduction the reconstitution of laurate co-hydroxylase activ­ state. Thus, the reduction state can be examined ity from a detergent solubilized preparation by measuring changes in the visible absorbance of cytochrome P450, NADPH cytochrome c range (Figure 4.2). This unique property stimu­ reductase^ ^' ^^, and a heat stable component, which lated research into the redox properties'^' '^, of the was later identified as the phospholipid phos­ enzyme and the complex processes of hydride/ phatidylcholine^^. electron transfer from NADPH, across the flavins The initial difficulties in identifying a physio­ and on to P450, discussed later in this chapter. logical role for CPR were due to the purification The cDNA and corresponding primary amino methods used, which incorporated trypsin or lipase acid sequences of several CPRs including rat'^, treatment^' ^. These resulted in the cleavage of rabbit^^, and human^' were obtained by the the amino-terminal membrane anchoring domain, mid-1980s, and the development of Escherichia which constitutes the first 60 or so amino acid coli expression systems paved the way for detailed residues and is responsible for interactions with the molecular characterization of the polypeptide phospholipid bilayer and P450^'^. Thus, while through site-directed mutagenesis. The three- proteolytically cleaved CPR is fully functional and dimensional structure of rat CPR was determined capable of reducing c3^ochrome c and a range of by X-ray crystallography in 1997 by Kim and artificial electron accepting compounds, it is unable coworkers^^, providing the structural prototype for to reconstitute with P450s. The intact form of the dual flavin oxidoreductases. reductase was eventually purified from liver micro­ somes using detergent solubilization procedures 2.2. The Diflavin Reductase and found to have a molecular weight of 76-80 kDa Family and to support P450-dependent reactions^^' ^^. In the early 1970s, the enzyme was shown to CPR is the prototype for a small family of contain one molecule each of FMN and FAD^^' ^^. diflavin reductases which are believed to have 300 400 500 600 700 Wavelength (nm) Figure 4.2. Absorbance spectra of oxidized and reduced human CPR. The absorption maxima for the oxidized enzyme are located at 380 nm and 454 nm. The direction of the arrow shows the absorption changes that occur upon reducton of the flavins, in this case using a 2-fold and 20-fold excess of NADPH. 118 Mark J.I. Paine et al. ferredoxin reductase | flavodoxin I FMN CPR IMS BM3 iPSgf^: NOS[ ':^1^^''- Figure 4.3. Schematic outline of the diflavin reductase family. Members contain an N-terminal FMN-binding flavodoxin-like domain and a C-terminal FAD/NADPH-binding ferredoxin reductase-like domain, which contains an additional linker region. Shown are: CPR, which has an amino-terminal membrane anchor region (Anc); NRl (Novel reductase 1); MSR (methionine synthase reductase), which contains an additional interdomain sequence; P450 BM3, which is fused to a P450 domain; and NOS (nitric oxide synthases), which has linked to a heme-containing oxygenase domain that is structurally distinct from the P450s. evolved as a result of a gene fusion event between electrons to the P450 monooxygenase complex, ancestral FMN- and FAD-containing flavopro- albeit with greatly reduced efficiency^^. A num­ teins^^. Indeed, CPR is one of only four mam­ ber of other diflavin reductases have been dis­ malian enzymes that contain both FMN- and sected into functional units including: rat CPR^^, FAD-binding domains, the other three being BM3^'' ^^, NRl^^, and methionine synthase reduc­ methionine synthase reductase^"*, NRl^^, and the tase^"^. The ability to separate CPR and other nitric oxide synthases^^ (Figure 4.3). Bacterial diflavin reductases into their component parts has members of the family include the B. megaterium greatly facilitated structural and functional studies cytochrome P450 BM3^^ and the reductase subunit of these enzymes. of sulfite reductase^^. While these are all struc­ turally related, there exist some key differences. For instance, methionine synthase reductase and 2.3. CPR Genes NRl both contain the same domain organization as P450 reductase but lack the membrane anchoring Apart from plants, which contain multiple sequence, and are thus located in the cytosol. CPR genes^^'^^, most organisms contain a single Methionine synthase also has a much larger inter­ CPR gene. At the time of writing, a relatively domain linker than CPR, although the functional modest total of 20 CPR genes have been identi­ significance of this is unclear. In nitric oxide syn­ fied, which will doubtlessly increase as a result of thase, the reductase region is fused with a heme the many genome sequencing projects currently domain, and has acquired an extra calmodulin- underway. New sequences can be monitored using binding domain to regulate electron transfer. the website www.icgeb.trieste.it,