Adrenodoxin reductase

In mitochondria (Fig. lb), the electron acceptor protein is also a flavoprotein termed NADPH-adrenodoxin reductase (MW 50 kDa) because it was discovered in the adrenal cortex and because it donates its electrons not directly to the P450 but to the smaller redox protein adrenodoxin (MW 12.5 kDa). The two iron-sulphur clusters of this protein serve as electron shuttle between the flavoprotein and the mitochondrial P450. 

Mitochondrial inner membrane of pig and rabbit liver (Axen et al., 1994). Recombinant CYP27 requires adrenodoxin and adrenodoxin reductase (Pikuleva etal, 1997). 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

Cytochromes P-450 may be found in other organelles as well as the SER including the rough endoplasmic reticulum and nuclear membrane. In the adrenal gland, it is also found in the mitochondria, although here adrenodoxin and adrenodoxin reductase are additional requirements in the overall system. Although the liver has the highest concentration of the enzyme, cytochromes P-450 are found in most, if not all, tissues. 

NADPH NADPH-adrenodoxin reductase (fp), adrenodoxin (Fe-S) P-450 Adrenal mitochondrial steroid hydroxylase 36-39)... 

FIGURE 21-47 Side-chain cleavage in the synthesis of steroid hormones. Cytochrome P-450 acts as electron carrier in this mixed-function oxidase system that oxidizes adjacent carbons. The process also requires the electron-transferring proteins adrenodoxin and adrenodoxin reductase. This system for cleaving side chains is found in mitochondria of the adrenal cortex, where active steroid production occurs. Pregnenolone is the precursor of all other steroid hormones (see Fig. 21-46). 

An example is adrenodoxin reductase (see chapter banner, p. 764), which passes electrons from NADPH to cytochrome P450 via the small redox protein adrenodoxin. This system functions in steroid biosynthesis as is indicated in Fig. 22-7.209a b Other flavin-dependent reductases have protective functions catalyzing the reduction of ascorbic acid radicals,210 211 toxic quinones,212-214 and peroxides.215-218... 

Once cholesterol is transferred to the inner mitochondrial membrane of steroidogenic tissues such as adrenals, ovaries and testes, it encounters the enzyme system known as the cholesterol SCC system. This probably comprises 20- and 22-hydroxylases and a C-20,22-lyase, all tightly bound to the inner face of the membrane and associated with a specific cytochrome />-450scc. In addition, molecular 02 is necessary together with NADPH reductase and non-haem iron sulphur protein, which are called adrenodoxin reductase and adrenodoxin, respectively, in the adrenal [24] (Fig. 3). 

Fig. 3. Cyclic reduction and oxidation of cytochrome P-450 in adrenal cortical mitochondria. FP. adrenodoxin reductase non-haem iron protein, adrenodoxin SH, substrate SOH. hydroxylated product (from Ref. 24, with permission).

The ability to form a stable one-electron-reduced radical (semiquinone) allows flavin cofactors to sit at the crossroads of two-electron and one-electron transfer chains. That is, they can be reduced by organic substrates two electrons at a time and be reoxidized by either obligate one-electron acceptors such as cytochromes (e.g., yeast cytochrome b2 or cytochrome b5 reductase/cytochrome b5) and iron-sulfur cluster proteins (adrenodoxin reductase/adrenodoxin) or by facultative one-electron acceptors such as benzoquinones (coenzyme... 

The spectra of adrenodoxin and testis iron protein reduced either by dithionite or by NADPH plus adrenodoxin reductase (flavoprotein) exhibit a distinct feature (Fig. 2) the absorption maxima at 455 mp, 414 mp, and 320 mp graetly decrease, although they do not disappear completely. A new distinct maximum appears at 540 mp. However, adrenodoxin can not be reduced by ascorbate or borohydride. The chemical reduction by an excess amount of dithionite gives an 86% decrease in the absorbance, whereas the enzymatic reduction produces a 65% decrease. This difference can be interpreted as gradual loss of iron from the chromophore upon reduction by dithionite. When the reduced form is reoxidized by air, the original spectrum can be regained. The identical characteristic is observed in preparations of testis iron protein. Therefore, adrenal and testis non-heme iron proteins are autoxidizable as well as bacterial and spinach ferredoxins are. 

Dramatic change in the ORD occurs upon enzymatic reduction with NADPH and adrenodoxin reductase under anaerobic conditions as shown in Fig. 4. The dispersion displays a fairly plain pattern in the visible range, although the peak of the principal effect appears to shift to shorter wave length, whereas the peak near 350 mp is not affected. 

Fig. 4. Optical rotatory dispersion of adrenodoxin (29). Curve A is the oxidized form and curve B is the enzymatically reduced form by NADPH2 and adrenodoxin reductase. Curve C is the reoxidized form (by the introduction of air)...

When adrenodoxin is reduced by NADPH and adrenodoxin reductase or by dithionite, a prominent signal appears as given in Fig. 8. A similar spectrum is obtained in the reduced testis protein (Fig. 8). The g-values of this type of signal are listed with reference to other non-heme iron proteins in Table 7. All of these non-heme iron proteins except rubredoxin... 

The oxidation-reduction potential (E6) of adrenodoxin is 164 mV at pH 7.4 and 26° C by the potentiometric titration method with dithionite in a nitrogen atmosphere (29). However, Estabrook and his colleagues obtain a value of —196 mV in the determination of the potential anaerobically by titration with NADPH plus adrenodoxin reductase using dyes with electromotive activity. The difference in value between the two... 

The number of electrons transferred per mole of adrenodoxin (n-value), determined from the slope of the potentiometric titration curve, is 2. This result is further supported by anaerobic titration of the protein with NADPH in the presence of adrenodoxin reductase (30). The result shows that one mole of NADPH is required to reduce one mole of adrenodoxin. Therefore, it is concluded that adrenodoxin transfers two electrons per mole of the protein. Obviously, this conclusion is incompatible with the EPR data which favor the interpretation that one of the two irons is reduced. This discrepancy can not presently be understood in detail. 

Recently the rat 24-OHase cDNA was recombinantly overexpressed in a bacterial cell line and reconstituted with its required cofactors [225]. Cell fractionation revealed the 24-OHase to be primarily localized in membranes, with minimum inclusion body formation, and that upon reconstitution with adrenodoxin, adrenodoxin reductase, and NADPH, 24-OHase activity was apparent. Interestingly, this preparation produced three... 

Figure 9.85 Normal phase HPLC profiles of the reaction product of the cholesterol side chain cleavage system. Peaks were identified on the basis of their retention times. (i4) Without cholesterol oxidase treatment. Cholesterol (100 nmol) was incubated with cytochrome P450scc (70 pmol) in the presence of adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Monitoring was at 214 nm. Peaks 1, cholesterol 2, pregnenolone 3, deoxycorticosterone acetate (internal standard) (B) The reaction mixture of (A) was further incubated with cholesterol oxidase at 37°C for 10 minutes. Monitoring was at 240 nm. Peaks 1, cholestenone 2, progesterone 3, deoxycorticosterone acetate (internal standard). (From Sugano et al., 1989.)...

NADH-cytochrome f>o reductase (9) and NADPH-cytochrome P-450 reductase (10, 11) are microsomal enzymes. The latter has been referred to until very recently as NADPH-cytochrome c reductase, since that is how it is assayed, but there is no cytochrome c in microsomes and its physiological acceptor seems to be cytochrome P-460. It is thus distinguished from NADH-putidaredoxin reductase (12), NADPH-adrenodoxin reductase (13), and NADH-rubredoxin reductase (14). The adrenodoxin reductase and the rubredoxin reductase, together with their respective iron-sulfur protein acceptors, each constitute a cytochrome P-460 reductase system. 

The crystal structure of NADPH-cytochrome P450 reductase (CPR), the common electron-transfer protein of Class 11 eukaryotic P450 systems, was reported in 1997. This was followed by the structures of adrenodoxin reductase (AdR) and adrenodoxin (Adx), the two electron-transfer proteins of the Class 1 mitochondrial P450 system. The crystal structure of a cross-linked AdR-Adx complex has also been reported. " Putidaredoxin reductase (PdR) and putidaredoxin (Pd) of the P450cam system have also been structurally characterized. ... 

Figure 8 The structures of the FAD-containing adrenodoxin reductase (AdR) and the Fe2S2 ferredoxin adrenodoxin (Adx) cross-linked by the side chains of Lys27 of AdR and Asp39 of Adx. The distance of closest approach of the two redox centers is...

Cytochromes are used in the conversion of cholesterol to the steroid hormones. Iliese hormones include aldosterone, cortisol, and the sex hormones. Synthesis of aldosterone, for example, occurs in the mitochondria of the adrenal cortex. One of the steps in aidostero ne synthesis is a hydroxylation that is cataly .ed by a complex of adrenodoxin reductase (FAD-containing protein), adrenodoxin (nonheme iron protein), and cytochrome P450 (heme protein). 

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