Adrenodoxin Reductase and Adrenodoxin MECHANISMS OF REDUCTION OF FERRICYANIDE AND CYTOCHROME

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...

in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these l-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and X-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction.
For cytochrome c reduction, the adrenodoxin reductase . adrenodoxin 1:l complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251,[7545][7546][7547][7548][7549][7550]. Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k = 4.6 s-l), but only slowly with a second cytochrome c UC = 0.1 to 0.3 s-l). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase . adrenodoxin complex appears to cycle between l-and 3-electron reduced states, via an intermediate 2-electroncontaining form produced by reoxidation by cytochrome (Fig. 10).
For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center.
$ These studies were carried out in part during the tenure of an Insurance Medical Scientist Scholarship from Prudential Insurance co. NADP+ remains bound to the 3-and 2-electron-reduced forms of the complex during catalysis, but can dissociate from the l-electron-containing form, allowing reduction of the complex by the next molecule of NADPH.
NADP+ is a competitive inhibitor to NADPH in adrenodoxin reductase-catalyzed ferricyanide reduction (K, = 24 PM), but shows a "mixed" pattern of inhibition of the adrenodoxin-dependent cytochrome c reduction. Binding of NADP+ to a low affinity binding site on adrenodoxin reductase (K,,,, = 200 PM), (a site different from the higher affinity site for reduction by NADPH) is shown spectrophotometrically to alter the interaction between adrenodoxin reductase and adrenodoxin.
This can account for the noncompetitive component of NADP+ inhibition observed for cytochrome c reduction.
Adrenodoxin reductase (EC 1.6.7.11, a single subunit, mono-FAD-containing enzyme, and adrenodoxin (adrenal ferredoxin), an iron sulfur protein of the ferredoxin type, function as an electron transport chain from NADPH to cytochrome P-450 in the adrenal cortex mitochondrial steroid-hydroxylation systems (cholesterol side chain cleavage, 11/3 hydroxylation, and 18 hydroxylation) (l-5). The flavoprotein can accept 2 electrons from NADPH with the concomitant production of a long wavelength-absorbing (6) NADP+-reduced flavoprotein charge transfer complex (7,8) and can transfer reducing equivalents from NADPH to hemoprotein, either the natural acceptor cytochrome P-450, or cytochrome c. Hemoprotein reduction requires the presence of adrenodoxin, a l-electron carrier (1, [9][10][11][12]. Adrenodoxin and adrenodoxin reductase have been shown to form a tightly associated 1:l complex when both components are in the oxidized form (12). We have previously shown this complex to be the catalytically active species for cytochrome c reduction and have demonstrated that at low ionic strengths the complex remains tightly associated, regardless of the observable oxidation states of either the flavoprotein or the ironsulfur protein (11,13). Thus, the complex should remain associated throughout a catalytic cycle. Our previous studies used equilibrium methods to demonstrate and investigate 2-and 3-electron-reduced states of the flavoprotein . iron-sulfur protein complex. Reduction of the protein. protein complex with NADPH was shown to produce a 1:l:l ternary complex of NADP+ adrenodoxin reduc-tase adrenodoxin, which contained 2 electrons assignable predominantly to the pyridine nucleotide-reduced flavoprotein charge transfer region (7,11). However, such studies did not demonstrate a l-electron-containing form of the protein protein complex. Since cytochrome c, the electron acceptor in these studies, can accept only a single electron, such a lelectron-containing species must be an obligatory product in the reoxidation of the 2-electron-containing complex by 1 eq of cytochrome c Present studies have utilized rapid mixing techniques to demonstrate a l-electron-containing form of the enzyme and have investigated its interaction with NADP+. These studies have also shown that during catalysis of cytochrome c reduction the adrenodoxin reductase-adrenodoxin system cycles between l-and 3-electron-reduced forms of the complex. A catalytic cycle for the flavoprotein-catalyzed ferricyanide reduction is also presented. Solutions were transferred anaerobically to a stopped flow loading syringe, using an apparatus consisting of a standard three-way metal syringe valve, one of whose ports was attached to a reservoir containing argon-bubbled anaerobic buffer. The second and third ports were connected to the stopped flow reservoir syringe and to a 20-gauge needle, respectively. Following flushing of the apparatus and syringe with anaerobic buffer, the needle was inserted into the serum cap of the tube containing the reduced adrenodoxin, and the contents were withdrawn into the stopped flow reservoir syringe. A second needle attached to the argon gas line (1 atm) was also in-  Oxidation-reduction potentials were calculated from the Nernst equation, as described previously (7, 11). Initial rates of cytochrome c and K,FeCN, reduction were measured at 550 nm and 420 nm, respectively, using t = 19,100 Mm' cm-' (16), and E = 1,020 M-' cm-' (17).
Spectra were recorded using a Cary 14 recording spectrophotometer.

Semiquinone
Form of Adrenodoxin Reductase -We have previously observed a spectrum resembling that of the "blue" or neutral form of flavin semiquinone, during air reoxidation of adrenodoxin reductase which had been reduced with an excess of NADPH (7). To demonstrate that this spectrum did indeed reflect a l-electron-containing species, we mixed adrenodoxin reductase with equimolar (NADPH plus K,Fe(C!N),J' in the stopped flow apparatus. Since NADPH donates two reducing equivalents to the flavoprotein, and K,Fe(CN), equimolar to flavoprotein should accept a single electron from many of the reduced flavoprotein molecules, many of the flavins should end up in the l-electron-containing form. Fig. 1, dotted line, shows the spectrum constructed from six stopped flow experiments at various wavelengths, plotting final absorbance at the end of 100 ms as a function of wavelength. A spectrum typical of neutral semiquinone is produced and may be compared to the spectrum of oxidized flavoprotein in this region ( Fig. 1, solid line). Residual absorbance at 700 nm suggests that in addition to the l-electron-containing form, a signifkant amount of 2-electron-reduced flavoprotein in charge-transfer association with NADP+ (7) remains. Since absorbance of this complex is flat from 500 to 700 nm, its presence does not interfere with the observation of the characteristic spectrum of the neutral flavin semiquinone.    Fig. 4. The amounts of cytochrome c reduced in rapid and slow phases were approximately equal, and the total amount reduced was 93% of the available cytochrome (see Table III). Reduction of the second cytochrome c by the l-electron-containing form of the complex is far too slow to account for the observed rate of turnover in catalytic experiments.
Therefore, a mechanism in which adrenodoxin reductase-adrenodoxin is first reduced by an NADPH and then reoxidized by two successive reductions of cytochrome c (i.e. cycling of the complex between fully oxidized and a-electronreduced), is ruled out.
However, when a second equivalent of NADPH is included along with the second cytochrome c (  Fig. 4 show that 75% of the 550 nm absorbance change occurs in the rapid phase. The total absorbance change represents 93% of the theoretically reducible cytochrome c. Thus, in turnover, the adrenodoxin reductase adrenodoxin complex must be cycling between the l-electron-reduced and 3-electronreduced forms.

Catalytic Properties of Adrenodoxin
Reductase and Adrenodoxin -Adrenodoxin reductase catalyzes the NADPH-dependent reduction of K,Fe(CN),. The double reciprocal plot of ferricyanide reductase activity versus NADPH concentration at four different NADP+ concentrations is shown in Fig. 5. The data indicate that the inhibition by NADP+ is competitive with NADPH. Although a K, for NADPH could not be determined directly from the line in Fig. 5 at zero NADP+, a plot of the apparent K,, values (Kubsereed), taken from the x-intercepts at various NADP+ concentrations in Fig. 5, uersus NADP+ concentration allowed determination of the K, (see Fig. 6). A K,n for NADPH of 2.6 PM and a K, for NADP+ of 24.0 pM are determined from the x-and y-intercepts of this plot according to the following equation.
Adrenodoxin stoichiometric to adrenodoxin reductase has no effect on either the rate of ferricyanide reduction or the pattern of inhibition by NADP+ for this reaction. The steady state kinetics of cytochrome c reduction by adrenodoxin reductase plus adrenodoxin, have been investigated previously (12): a K,,, of 1.8 pM for NADPH was determined, and a double reciprocal plot of velocity of cytochrome c reduction versus NADPH concentration, with varied NADP+ concentration, showed a pattern of "mixed" (12) inhibition by NADP+. We have confirmed this result; the lines at zero NADP+ and at four different NADP+ concentrations intersect to the left of the y-axis and above the x-axis. A K,,, for NADPH of 2.0 pM was determined, in good agreement with the results of Chu and Kimura.

Binding of NADP+ to Adrenodoxin
Reductase .Adrenodoxin Complex -The observation of "mixed" inhibition by NADP+ in cytochrome c reduction and "pure competitive" inhibition in ferricyanide reduction suggested to us that the difference between the two patterns could reflect the requirement for adrenodoxin in the cytochrome c but not the ferricyanide reaction. We therefore sought possible optical changes which could reflect an effect of NADP+ on adrenodoxin-adrenodoxin reductase interactions. NADP+ binding has been shown to induce a perturbation of the spectrum of adrenodoxin reductase (18  5 (left). Inhibition by NADP+ of NADPH-ferricyanide reductase activity.
Plot of l/u versus l/(NADPH) with the indicated concentrations of NADP+, with u expressed as the a-electron turnover (min-I) of 16.7 nM adrenodoxin reductase. FIG. 6 (right).
Apparent K, values were determined from extrapolation of lines in Fig.   5 to their x-intercepts. spectrum produced is shown in Fig. 7, following addition of 0.45 (Fig. 7A, dashed line) or 0.90 (Fig. 7A, solid line) equivalents of NADP+ to the flavoprotein.iron-sulfur protein complex. An isosbestic point is seen at 482 nm, with a peak at 498 nm and decreased absorbance at wavelengths below 482 nm. However, on addition of NADP+ above 1.4 eq (Fig. 7A, other curves), isosbesticity is lost, and a new absorbance peak is seen at 392 nm. These data demonstrate sequential formation of at least two different species on titration of the protein. protein complex with NADP+, suggesting at least two different binding sites (with different binding constants) for pyridine nucleotides. The difference spectrum due to binding to the high affinity binding site is seen on addition of a less than stoichiometric amount of NADP+ to the protein. protein complex (Fig. 7B, dashed line). The difference spectrum produced on binding to the low affinity site was calculated by subtraction of the difference spectrum at 4.55 eq from that at 13.63 eq of NADP+ (Fig. 7B Binding to the high affinity site produces no long wavelength absorbance changes (beyond 540 nm) either in the presence or absence of adreno-DISCUSSION doxin. However, binding of NADP+ to the low affinity binding site produces longer wavelength absorbance changes when adrenodoxin is present, but not when it is absent. Because adrenodoxin, but not adrenodoxin reductase absorbs in this region, these data indicate that the binding of NADP+ to the low affinity site alters the flavoprotein-iron sulfur protein interaction in some way. This alteration does not involve dissociation of the complex, since the characteristic difference spectrum for dissociation of this complex (10) is not produced. Dissociation of the complex would be expected to produce a decrease rather than the observed increase in the absorbance bevond 540 nm. In addition. this absorbance cannot be due to flavin semiquinone or charge transfer complex, since all components are fully oxidized. reduction of adrenodoxin reductase, formed rapidly, is the charge transfer complex, in which the NADP+ is tightly bound (K, = 1 x lo-" M) to the reduced flavoprotein (7). Dissociation of NADP+ then occurs at some step following the reduction of the first ferricyanide. The sequence of dissociation of NADP+ uersus oxidation by a second K,,Fe(CN),, is undefined in this proposed mechanism, since rates for these processes are unknown. The catalytic cycle of adrenodoxin reductase with The concentration of adrenodoxin-adrenodoxin reductase with NADP+ bound to the high and low affinity sites may be determined from the absorbance changes at 487 nm and 482 nm, respectively, with extinction coefficients for each of these species determined by extrapolation of the absorbance change at each wavelength to infinite NADP+ concentration. The titration of the complex with NADP+ followed at these wavelengths and at 560 nm is shown in Fin. 8. Two clearlv distinguishable binding processes were seen to be associated with 487 nm and 482 nm absorbance changes. Long wavelength absorbance changes (560 nm) are shown in Fig. 8 to have the same NADP+ concentration dependence as the 482 nm changes. No binding to the low affinity site is seen until a greater than stoichiometric amount of NADP+ is added, as indicated by the lag period preceding absorbance changes at 560 and 482 nm. Assuming that binding of one NADP+ to each of the two binding sites produces each of the observed difference absorption spectra, the concentration of unbound NADP+ at any NADP+ enzyme complex ratio can be calculated, and from this, dissociation constants for binding of NADP+ to the high and low affinity binding sites on the flavoprotein. These constants were calculated from the data obtained at 1 and 10 eq of NADP+ per mol of enzyme complex; the values were 13 and 200 PM, respectively. We had previously demonstrated by ultrafiltration binding studies (7) a high affinity site for binding of one NADP+ to adrenodoxin reductase, (K,, = 14 PM), in agreement with present studies. The earlier studies could not ferricyanide (Fig. 91, is reminiscent of the cytochrome 6, reductase system (22), in which a one-FAD enzyme, probably in charge transfer complex with NAD(H), reduces 2 mol of a lelectron acceptor (cytochrome b, or ferricyanide) in two successive steps, using both the fully reduced flavin and the semiquinone as reductants.
Competitive inhibition of NADPH binding by NADP+ is permitted by this proposed mechanism. The observed K, of 23 PM compares reasonably well with the observed KS (13 to 14 PM) described in the present and previous (7) studies. Neither the rate of K,,Fe(CN), reduction, nor the pattern of inhibition by NADP+ is affected by the presence of adrenodoxin stoichiometric to adrenodoxin reductase, suggesting that when present, the iron-sulfur center does not participate in the reduction of K,Fe(CN),,. This suggestion is further supported by the second order rate constant of 1.8 x 10"'s~' Mm" for reduction of K,Fe(CN),, by reduced adrenodoxin.
At catalytic concentrations of adrenodoxin, this could account for a rate of ferricyanide reduction on the order of only 0.01 s'. Thus, adrenodoxin does not alter the mechanism of NADPH-ferricyanide oxidation-reduction by adrenodoxin reductase and should not participate in this reaction.
Catalysis of NADPH-cytochrome c reduction, however, requires the presence of both adrenodoxin reductase and adrenodoxin, in a 1:l ratio (11). The proposed mechanism for the cytochrome c reductase activity of the adrenodoxin reductase.adrenodoxin complex is summarized in Fig. 10. The complex first reacts with NADPH with an apparent first order rate constant of 18 ss', to produce the NADP+-AK-ADX species, a form which contains 2 electrons in a reduced flavin NADP' charge transfer complex of low dissociation constant (1 x lo-* M) (7  [9][10][11][12]. Second, reduced adrenodoxin reacts rapidly (k > 300 s-l) to reduce cytochrome c, in an oxygen independent reaction (present studies).
Third, reaction of NADPH with equimolar enzyme complex, in the presence of an equivalent of cytochrome c causes relatively rapid (h = 4.6 s-l) reduction of the cytochrome c (see Table III). Thus, we conclude that in order for the 2-electron-reduced complex to reduce cytochrome c, one of the electrons must reside in the iron-sulfur center.
In addition, thermodynamic considerations support the existence of such a species. The free energy change (AGJ for Reaction C in Fig. 10 (Fig. 10, Reaction D). Fig. 11 also demonstrates that reduction of the first cytochrome c provides sufficient energy to "pull" Reaction C, Fig. 10  This uncertainty is signified by the question murk in Fig. 11.
Reactions C plus D in Fig.  10 proceed with an overall apparent first order rate of 4.6 s-l (see Table III), near the rate expected for the rate limiting step for the catalytic cycle (turnover = 3.2 5-l). As shown in Table  III Fig. 10.
The l-electron-containing species is shown to be but slowly reactive with cytochrome c (k = 0.11 to 0.33 s-l). This lack of reactivity is not due to unfavorable free energy of reaction, since essentially all the cytochrome c is reduced in Experiment 2,  Fig. 10). The mechanism proposed for cytochrome c reduction (Fig.  lo), when considered alone, predicts competitive inhibition by NADP+ when NADPH is varied." However, catalytic studies demonstrate a "mixed" pattern of inhibition by NADP+, indicating not only a competitive component but also a noncompetitive component of NADP+ inhibition.
The present studies show that the latter component is observed in cytochrome c, but not ferricyanide, reduction. Chu and Kimura have previously suggested a second pyridine nucleotide-binding site to account for this inhibition (8). This has been directly demonstrated in the present studies: at least two binding sites for NADP+ exist on the flavoprotein, as shown by the data of Figs. 7 and 8. The binding constant for the high affinity binding site (& = 13 PM) corresponds relatively closely to the Ki for NADP+ inhibition of adrenodoxin reductase-catalyzed NADPH-ferricyanide reductase activity (Ki = 24 FM). Since the latter adrenodoxin-independent activity demonstrates a competitive rather than a mixed pattern of inhibition by NADP+, the high affinity site must represent the site for reduction by NADPH of the FAD in adrenodoxin reductase. The low affinity site (Kd = 200 PM) is also on the flavoprotein, but appears to affect the flavoprotein-iron sulfur protein interaction (Fig. 7). Since only the adrenodoxin-dependent activity (cytochrome c reduction) demonstrates the noncompetitive component of inhibition by NADP+, we propose that the low affinity NADP+-binding site participates in the adrenodoxin reductase-adrenodoxin interaction. Cytochrome P-450, the natural hemoprotein electron acceptor, requires the input of 2 electrons per heme in order to carry out steroid hydroxylation.
Studies by Sligar et al. (25) have indicated that in electron transport to the bacterial cytochrome P-450,,, the 2 electrons are transferred in two sequential steps. Electron transport to cytochrome P-450 is similar to that for cytochrome c reduction, since it displays an absolute dependence for NADPH-supported activity on both adreno-6 The rate equation for a slightly simplified but kinetically equivalent version of this mechanism has been derived by us using the method of King and Altman (24). Lineweaver-Burk plots constructed using this equation show a pattern of competitive inhibition by NADP+ when NADPH is varied. I-NADP+ -I-AR-ADX + 2 CYT Cred doxin reductase and adrenodoxin (1). Thus, we suggest that electron transport to the membrane-bound adrenal mitochondrial cytochrome P-450 occurs by the mechanism shown in Fig.  10 for cytochrome c reduction, but with donation of 2 electrons in two consecutive l-electron transfers to a single cytochrome P-450, rather than to two different cytochrome cs.
Electron transport from NADPH to cytochrome P-450 in liver microsomes has been shown to proceed via an FAD-FMN flavoprotein (26-28), and does not require an iron-sulfur protein. Catalytic and mechanistic studies of hemoprotein (cytochrome c) reduction by liver enzyme reveal a number of similarities to hemoprotein reduction by the adrenodoxin reductase'adrenodoxin complex. First, two different oxidation-reduction centers (FAD and Fez-S,* in the adrenal system and FAD and FMN in the liver system) are involved in both reaction mechanisms. In addition, both reductase systems remain partially reduced during their catalytic cycles, the liver microsomal reductase cycling between 2-and 4-electronreduced (29, 30) or perhaps l-and 3-electron-reduced (26) states, and the adrenal mitochondrial reductase complex cycling between l-and 3-electron-reduced states, as shown in the present studies. The complex FAD-FMN-iron/sulfur hemoprotein, Escherichia coli sulfite reductase, also has its FAD-FMN pairs cycling between the l-electron and S-electron forms (31, 32).
The interaction of two flavin groups in flavoproteins has been proposed by Hemmerich (discussion of Ref. 32) to allow not only the splitting of an electron pair into single electrons, but also donation of each electron at constant oxidation-reduction potential. This function has been proposed for the FAD-FMN pair in sulfite reductase (32), and may function similarly in the microsomal cytochrome c (cytochrome P-450) reductase. Since adrenodoxin reductase contains only a single flavin, two sequential electron transfers from the reduced flavin group to ferricyanide must occur from electrons at two different oxidation potentials, i.e. those of the oxidized/semiquinone and semiquinone/reduced flavoprotein couples (approximately -360 mV and -230 mV, repectively). However, formation of the adrenodoxin reductase adrenodoxin complex provides a means by which 2 electrons from NADPH may be delivered sequentially at a constant potential (i.e. that of the iron-sulfur group) to the acceptor cytochrome c or cytochrome P-450.
Similar 1:l complexes between flavoproteins and iron-sulfur proteins have also been observed in NADH-rubredoxin reduc-tae and rubredoxin