BASES DE VALUACIÓN E INDEMNIZACIÓN DE DAÑO

For all CYPs, electrons are derived from NADPH and their transfer, crucial for the enzyme’s catalytic activity, is facilitated by cofactors.

Figure 3: Schematic illustration of the two different routes of electron transfer for mitochondrial type 1

and microsomal type 2 CYPs. For type 1 enzymes, electrons are transferred via adrenodoxin reductase (AdR) and adrenodoxin (Adx). Type 2 enzymes receive electrons via the flavoprotein P450 oxidoreductase (POR). The crystal structures of the indicated proteins are shown in this figure: for the CYP enzyme, the crystal structure of CYP2D6 was chosen as a representative example. Data were retrieved from the pdb online protein databank; PDB IDs: FDR: 1CJC; FDX1: 3P1M; POR: 3QFS; CYP2D6: 2F9Q.

Depending on the redox-cofactor system, which also determines their sub- cellular localization, CYPs are further sub-classified into two main groups: seven human CYPs are expressed in the mitochondria and are termed “type 1” enzymes. They receive electrons via the flavoprotein adrenodoxin (Adx; also ferredoxin), which is in turn reduced by its co-enzyme adrenodoxin reductase (AdR; also ferredoxin

NADPH NADP+ e- NADPH NADP+ e- e- e- e- AdR Adx POR CYP

type 1

type 2

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reductase); the other 50 “type 2” CYPs are found in the endoplasmic reticulum (ER) and electron transfer from NADPH is facilitated by the single flavoprotein P450 oxidoreductase (POR) (Figure 3).

1.1.3.1. Adrenodoxin (Adx) and Adrenodoxin reductase (AdR)

Adrenodoxin (Adx) and adrenodoxin reductase (AdR) are expressed in the mitochondrial matrix and inner mitochondrial membrane (IMM) of various tissues, in particular steroidogenic cells, where they support electron flux to type 1 CYPs.

Adx is a soluble iron-sulfur protein that is only loosely attached to the IMM and mainly resides freely within the mitochondrial matrix. AdR is a flavoprotein attached to the IMM containing a flavin adenine dinucleotide (FAD) in its core domain and binds NADPH at its N-terminal domain, which interacts with the FAD domain when ’activated’ by NADPH to allow electron transfer to the FAD. The FAD domain forms a positively charged ‘cleft’ docking to the negatively charged surface of the interaction domain of Adx, which is in proximity to the Fe2/S2 core of the core domain (Ziegler et

al., 1999). There is evidence that the same surface area that docks to the AdR protein also interacts with the positively charged co-factor binding site of the CYPs (Vickery, 1997).

1.1.3.2. P450 oxidoreductase (POR)

POR is a membrane-bound flavoprotein that supports electron transfer from NADPH to all microsomal (type 2) CYPs. Only three CYPs are involved in steroidogenesis, namely CYP17A1, CYP21A2 and CYP19A1 (see Table 1).

The wide tissue distribution of the POR protein indicates that it supports many biological processes, including electron transfer to all major drug-metabolising CYP enzymes, and it has been shown that POR not only supports electron transfer to

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CYPs, but also to other redox systems involved in crucial metabolic processes like squalene monooxygenase (Ono and Bloch, 1975), haeme oxygenase (Wilks et al., 1995), fatty acid elongase (Ilan et al., 1981) and post-translational modification of proteins involved in the hedgehog signalling pathway (Aguilar et al., 2009).

The structural and biochemical properties of POR have been studied extensively based on the rat crystal structure, which shares 94% amino acid identity with the human protein (Wang et al., 1997), but also the human POR protein has recently been crystallised (Xia et al., 2011). POR contains two derivatives of the nucleic acid riboflavin: a flavin adenine dinucleotide (FAD) and a flavin mononucleotide (FMN), which are located in major domains forming the two main lobes of the POR protein responsible for electron transfer. The N-terminal FMN domain is flexibly connected with the FAD domain by several α-helices forming the connecting domain, which brings the two riboflavin domains together once electrons are received by the C-terminal NADPH binding domain and transferred to the FAD moiety. A further 25 amino acid hinge region loosely connects the FMN with the connecting domain, suggesting a high flexibility in between these domains. NMR and x-ray crystallographic studies revealed drastic conformational changes during intra- molecular electron transfer: once electrons are received by the FAD domain, the two lobes (FAD and FMN domains) come close together, like wings of a butterfly, and the electrons are passed on from the FAD to the FMN domain (Ellis et al., 2009; Miller and Auchus, 2011). Subsequently, negative surface charges at the FAD domain ‘dock’ POR to positively charged basic residues of the co-factor binding site of the CYP protein to bring the FMN domain in close proximity to the CYP haeme centre, thereby passing on the electrons (Miller and Auchus, 2011).

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The POR gene is located on chromosome 7q11.2 and consists of 16 exons, of which the first one is not transcribed. The coding region is 2043 base pairs long and encodes for the 681 amino acid protein. The human POR gene is highly polymorphic and sequencing analysis in over 800 subjects from different ethnicities indicate a variety of sequence variants, some of which have some impact on CYP-mediated activities and cytochrome c reduction and NADPH oxidation (Huang et al., 2008). There does not seem to be a hotspot for polymorphisms or mutations within the gene structure of POR (Huang et al., 2008; Krone et al., 2007a; 2012).

1.1.3.3. Cytochrome b5 (CYB5A)

Cytochrome b5 (CYB5A) is a small (~ 16kDa) membrane-bound protein that contains a haeme molecule as its prosthetic group. A soluble isoform lacking the C- terminal membrane anchor is expressed in erythrocytes, the full protein containing the transmembrane domain and the haeme-domain is expressed in various tissues, but with high levels in liver, adrenals and gonads (Dharia et al., 2004; Suzuki et al., 2000).

CYB5A interacts with POR and CYP17A1 to further support electron flux from NADPH, in particular the higher demand for electrons required for its 17,20 lyase activity (see also section 1.1.8.1). However, the redox-potential between POR and CYB5A is unfavourable and hence makes it unlikely that CYB5A receives electrons in the context of CYP reduction, although its prosthetic group would be able to carry electrons. In addition, in vitro studies show that truncated CYB5A protein lacking the prosthetic group (apo-b5) stimulates the 17,20 lyase activity of CYP17A1 equally to holo-b5, suggesting that this co-factor supports allosteric interaction between POR and CYP17A1, rather than conducting the flux of electrons directly (Auchus et al.,

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In a broader context, CYB5A has been shown to support other physiological processes like fatty acid desaturases (Guillou et al., 2004) and other P450 monooxigenases (Schenkman and Jansson, 2003), including hepatic CYPs involved in steroid metabolism and drug detoxification (Yamazaki et al., 1996a; 1996b). Recent in vitro studies also suggest additional roles of CYB5A in steroidogenesis, in particular by enhancing HSD3B activity leading to an increase of androstenedione production (Goosen et al., 2013; 2011; Storbeck et al., 2013).