Adrenodoxin Interaction with Adrenodoxin Reductase and Cytochrome P-45OsCc

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRV Val. 259, No. 16, Issue of August 25, pp , by The American Society of Biological Chemists, Inc. Printed in U. S. A. Adrenodoxin Interaction with Adrenodoxin Reductase and Cytochrome P-45OsCc CROSS-LINKING OF PROTEIN COMPLEXES AND EFFECTS OF ADRENODOXIN MODIFICATION BY 1-ETHYL-3-(3-DIMETHYLAMINOPROPYL)CARBODIIMIDE* (Received for publication, February 21, 1984) J. David LambethS From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia Lois M. Geren and Francis Millett From the Department of Chemistry, University of Arkansas, Fayettville, Arkansas Modification of the three carboxyl groups on adren- cytochrome P-450.,,). - - odoxin using a water-soluble carbodiimide (l-ethyl-3- (3-dimethylaminopropyl)carbodiimide) caused a 2e- e- e- NADPH AR ADX * P-450 weakening of the binding of this iron-sulfur protein to both its electron donor protein, adrenodoxin reductase, Adrenodoxin, a one electron acceptor, is an unusually acidic and its electron acceptor protein, cytochrome P-450,. protein (11 aspartates and 7 glutamates per 114 amino acids Based upon the proximity of the modified groups, the total; Ref. 6), and has been shown to form tight 1:l complexes site on adrenodoxin for interaction with the other two with both adrenodoxin reductase (7,8) and with cytochromes proteins is likely to be either identical or highly over- P-450 (3, 9, 10). The strength of interaction for both comlapping, and formation of a ternary complex among plexes is reduced considerably at high ionic strength (ll), the proteins is precluded. Upon incubation of adreno- suggesting the contribution of electrostatic interactions to the doxin and either adrenodoxin reductase or cytochrome P-450 plus the carbodiimide (l:l), covalently crosslinked species were formed. When all three proteins were incubated with the cross-linker, only the binary complexes were formed, and no higher order (e.g. 1: 1: 1 complex of the flavoprotein, the iron-sulfur protein and the or 1:2:1) complexes were seen. These studies indicate cytochrome, formed in the presence of cholesterol and phosthat adrenodoxin forms exclusive binary complexes pholipid, and have suggested a role for such a complex in the with its electron transfer partner proteins, and thus catalytic cycle. In other studies from several laboratories, only provide a physical explanation for the proposed role evidence of for the two binary complexes was found, and no adrenodoxin as a mobile electron shuttle between NADPH-adrenodoxin reductase and cytochrome P- 450,,. Adrenal cortex mitochondria contain a monooxygenase enzyme system which carries out the three-step oxidative side chain cleavage of cholesterol, an NADPH-dependent reaction requiring a total of six electrons (3 mol of NADPH) plus three molecular oxygens/side chain cleaved (1, 2). Cytochrome P- 450,,, an integral inner membrane heme protein (3), binds both the substrate and molecular oxygen, and catalyzes the oxygen activation and three successive substrate oxidations which yield sequentially 22R-hydroxycholesterol, 20,22R-dihydroxycholesterol, and finally pregnenolone (2, 4). Two other protein components, the flavoprotein NADPH-adrenodoxin reductase and the iron-sulfur protein adrenodoxin, form a short electron transport chain which transfers electrons from NADPH to cytochrome P-450 (5), as shown below: (AR = adrenodoxin reductase, ADX = adrenodoxin, and P-450 = * This work was supported by National Institutes of Health Grants AM27373, GM 20488, BRSG 2 S07, and RR The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. j Recipient of Reseach Career Development Award AM01085 from the National Institutes of Health binding. Reports of the existence of 1:l:l or higher order complexes among the three proteins are contradictory. Kid0 and Kimura (12) have reported the existence of a ternary physical or kinetic evidence for higher order complexes was seen (Ref. 13 and references therein). We have provided thermodynamic and kinetic evidence indicating that adrenodoxin functions as a mobile electron shuttle (10, 11, 13, 14). According to this mechanism, adrenodoxin first binds to and accepts an electron from adrenodoxin reductase, then dissociates, and finally associates with and transfers its electron to cytochrome P-450; the reoxidized iron-sulfur protein then migrates back to the flavoprotein and the cycle is repeated. Both the reduction states of the proteins and the substrate binding to cytochrome P-450,, have been shown to regulate the protein-protein interactions which comprise the shuttle mechanism (10, 14). Since the binding of adrenodoxin to the flavoprotein appears to be competitive with its binding to the cytochrome (i.e. its association with one protein precludes its association with the second, (3, 15, 16)), we have proposed (13) that the same site (or overlapping sites) on adrenodoxin is (are) involved in the interaction with both adrenodoxin reductase and with cytochrome P-450. In the present study, we have used a water-soluble carbodiimide, EDC, to compare the interactions of adrenodoxin with both adrenodoxin reductase and cytochrome P-450,,. Such reagents have been shown to modify carboxyl groups only in highly negatively charged environments, thus leading The abbreviations used are: EDC, l-ethyl-3-(3-dimethylaminopropy1)carbodiimide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Mops, 4-rnorpholinepropanesulfonic acid; SDS, sodium dodecyl sulfate.

2 10026 Adrenodoxin Interaction to a selective modification of a limited number of aspartate and glutamate residues (17-20). For example, 4 residues are modified in cytochrome oxidase (18). In adrenodoxin, only three are modified (Glu 74, Asp 79, and Asp 86; Ref. 20). In the present studies we have extended earlier studies on the effects of adrenodoxin modification on the interaction with adrenodoxin reductase to now include effects on its binding to cytochrome P-450,,. In addition, we have utilized the ability of EDC to cross-link a carboxyl to a nearby amino group (20,21) to investigate further the nature of the possible charge-paired complexes of adrenodoxin with the flavoprotein and the cytochrome. EXPERIMENTAL PROCEDURES Materiak-NADP', cytochrome c (horse heart, type 111), glucose- 6-phosphate, glucose-6-phosphate dehydrogenase, Tween 80, dithiothreitol, EDTA, Hepes, and EDC were obtained from Sigma. Dimethylamine borane was purchased from Aldrich, and sodium dodecyl sulfate was from Bio-Rad. Methods-Adrenodoxin, adrenodoxin reductase, and cytochrome P-450, were purified as described previously (22, 23). Homogeneity was assessed by SDS-polyacrylamide gel electrophoresis. Adrenodoxin was &methylated quantitatively as described previously (20), using two additions of 60 mm dimethylamine borane and 80 mm formaldehyde to 5 mg/ml of the iron-sulfur protein in 0.2 M sodium phosphate, ph 7.0 (2 h at 25 "C). After removal of excess reagent on a small P-2 column, the sample was dialyzed and concentrated. In the resulting methyl adrenodoxin, all the amino groups were modified the derivative was fully active in the NADPH-cytochrome c reductase assay (20). Such derivatization does not change the net charge on the modified amino group. EDC modification was carried out by treating methyl adrenodoxin (20 pm) with 2 mm EDC at room temperature in 10 mm sodium phosphate, ph 7.0. The reaction was allowed to continue until only approximately 15-20% of the original activity was present in the NADPH-cytochrome c reductase assay; the reaction was then quenched with 0.1 M ammonium acetate. The assay was carried out in 50 mm sodium phosphate, ph 7.0, containing 66 nm adrenodoxin reductase, 190 nm adrenodoxin (or derivative), 27 p~ cytochrome c, 12 p~ NADP', 307 PM glucose-6-phosphate, and 0.1 unit/ml of glucose-6-phosphate dehydrogenase. Methylated rather than native adrenodoxin was used in these studies to prevent intra-polypeptide cross-linking between carboxyl- and amino-containing residues. This modification produces a positive rather than a negative charge on the modified group. Cross-linking between proteins was carried out by incubating adrenodoxin with either adrenodoxin reductase, cytochrome P-450, or both proteins, in the presence of EDC (20). The reaction was carried out at room temperature with slow stirring in 5 mm Mops buffer, ph 6.5, with 10% glycerol. Other incubation conditions were also tested (e.g. detergent or phospholipid plus cholesterol); the above conditions, however, provided the greatest yield. Aliquots were taken both before EDC addition, and at 1, 3, and 6 h. Further incubation did not increase the yield of cross-linked product. At time zero, freshly dissolved EDC (1 mm final) was added, and fresh EDC (an additional 1 mm) was added following removal of each aliquot to replenish any hydrolyzed reagent (total EDC added was 4 mm at the last time point). Aliquots (20 pl) were added to 20 pl of dissociation buffer (1% SDS, 1% mercaptoethanol, 400 mm Napi, ph 3.0), mixed by vortexing, and frozen for subsequent analysis by SDS-polyacrylamide gel electrophoresis as described by Rudolph and Krueger (24). Approximately 20 pg of protein were used for each gel lane. All optical spectra were recorded using a Varian 219 spectrohpotometer. Binding of adrenodoxin to cytochrome P-450 was monitored by following changes in the Soret maximum of the enzyme at 418 nm (low spin) and 392 nm (high spin heme). Since absorbance changes reflect the enhanced binding of cholesterol upon adrenodoxin binding (IO), cholesterol was included as indicated. Kd and K,,, values provided in the table were calculated using a weighted nonlinear regression analysis, using a computer program adapted from Duggleby (25). Such a direct fit to the data avoids distortions present in most linear fitting methods. For ease of qualitative interpretation, data are displayed on Lineweaver-Burk plots. with Reductase Cytochrome and P-450,, RESULTS Effect of Adrenodoxin Modification on NADPH-Cytochrome c Reductase Actiuity-NADPH-cytochome c reductase activity was measured as a function of the concentration of either native adrenodoxin, methyl adrenodoxin, or EDC-modified methyl adrenodoxin (see Fig. 1). Methylation had no effect on the kinetic parameters, but, as shown previously (20), EDC-modification of carboxyl groups (Glu 74, Asp 79, and Asp 86) increased the K, for adrenodoxin by a factor of 4 to 9 in two preparations of the derivative. No effect on the V, was seen, Table I summarizes the kinetic parameters for the native and modified adrenodoxins, using the two preparations. The two EDC-methylated adrenodoxin preparations differed in the extent of modification of the carboxyl groups at Glu 74, Asp 79, and Asp 86. In preparation a, each of these carboxyls was labeled to an extent of about 60%, while in preparation b, each was about 75% labeled based on high pressure liquid chromatography peptide maps as described l/cadxi (rm FIG. 1. Effect of adrenodoxin modification with EDC on NADPH-cytochrome c reductase activity. Incubations were carried out in 100 mm NaC1, 0.1 mm EDTA, 20 mm Hepes buffer, ph 7.2, with adrenodoxin reductase (14nM), cytochrome c (27 PM), NADP' (12 PM), glucose-6-phosphate dehydrogenase (0.2 units/ml), plus the indicated concentrations of either native (a), methyl adrenodoxin (0), or the EDC-derivative of methyl adrenodoxin (a). Glucose-6-phosphate (500 p~ final) was added to initiate the reaction. Cytochrome c reduction was monitored by the increase in 550-nm absorbance. TABLE I Effect of amino acid modification on the binding properties of adrenodonin a and b refer, respectively, to two different preparations of adrenodoxin and its derivatives. K, for adreno- Kd or cyto- Derivative doxin reductase chrome P-450, Native adrenodoxin (a) / /- 0.1 (b) / /- 0.01" Methylated adrenodoxin (a) 3.56 n.d. +/- 0.7 (b) / EDC-methylated (a) / /- 8.0 adrenodoxin (b) / /- 4.0" In the second series of experiments, the cholesterol concentration was doubled, from 26 to 52 p ~ in, order to increase the strength of adrenodoxin binding (lo), and to increase the observed absorbance changes, thus reducing the standard error.

3 Adrenodoxin Interaction with Reductase and Cytochrome P-450,, previously (20). Thus, modification of Glu 74, Asp 79, and Asp 86 significantly increased the K, for adrenodoxin in the NADPH-cytochrome c reductase assay. The effect was on the adrenodoxin-adrenodoxin reductase interaction rather than the adrenodoxin-cytochrome c interaction, since cytochrome c was saturating. We have previously shown (1 1) that the rate constants for electron transfer in this assay are such that the derived K, is approximately equal to the Kd value for the adrenodoxin-adrenodoxin reductase complex. As discussed previously (20), the fact that the apparent V, was not affected does not necessarily mean that the V, of the individual derivatives was not affected. The preparations contain amixture of derivatives, and also possibly somenative adrenodoxin which could lead to a normal V,,, at sufficiently high concentrations. Effect of Adrenodoxin Modification on Adrenodoxin-Cytochrome P-450,, Interactions-Binding was measured using the adrenodoxin-induced high spin optical changes in the cytochrome heme (see Experimental Procedures ). Fig. 2 shows plots of the inverse of the absorbance changes versus the inverse of the adrenodoxin concentration. Methylation did not change the Kd for binding, but did increase the maximum extent of spectrophotometric changes by a small but reproducible amount. Modification of carboxyl groups with EDC raised the Kd by a factor of 5 to 7 in the two preparations (see Table I), compared with the corresponding native or methylated adrenodoxin. Cross-linking of Native Adrenodoxin to Adrenodoxin Reductase and Cytochrome P-450, The water-soluble carbodiimide EDC has proven useful for the cross-linking of chargepaired protein-protein complexes such as adrenodoxin-cytochrome c (20) and cytochrome c-cytochrome c oxidase (21). In the present studies, we have demonstrated the facile formation of cross-linked complexes between adrenodoxin and both adrenodoxin reductase and cytochrome P-450,. Fig. 3, lanes 1-4, shows the time course for production of the higher molecularweight cross-link of adrenodoxin reductase and adrenodoxin; lanes 5-8 demonstrate the time course for formation of the adrenodoxin-cytochrome P-450 complex. No further yield of cross-linked species was seen in either case upon longer incubations. Studies were carried out initially with 1:1 ratios of both components (not shown). In both cases the yield of cross- 100 c m / a5 l/cadxl 1.0 py FIG.2. Effect ofadrenodoxin modification with EDC on the binding of adrenodoxin to cytochrome P-450,. Titrations were carried out in 20 mm Hepes buffer, ph 7.2, containing 0.1% Tween 80, 26 p~ cholesterol, 0.1 mm EDTA, 0.1 mm dithiothreitol, and 100 mm NaCI. Cytochrome P-450,(0.5 p ~ was ) titrated with either native adrenodoxin (O),methyl adrenodoxin (01,or the EDC-derivative of methyl adrenodoxin (B).Absorbance a t 418 and 392 nm was corrected for dilution, and used in calculations as described under Experimental Procedures ADX AR-ADX AR P450 ADX ADX FIG.3. Formation of covalent cross-linked speciesof adrenodoxin-adrenodoxin reductase and adrenodoxin-cytochrome P-450,. Shown are SDS-polyacrylamide gels (10%)of adrenodoxin ( A D X ) (50 p ~ incubated ) with either adrenodoxin reductase (AR) (20 p ~ lanes, 1-4) or cytochrome P-450, (20 p M, hnes 5-8), plus EDC (1mM final), as described under Experimental Procedures. In each case, the first lane shows the proteins prior to addition of EDC, and the subsequent lanes represent the 1-,3-, and 6-h time points, respectively. linked species was relatively low, presumably due to the competing hydrolytic reaction which can follow the initial reaction with EDC (17). To obtain higher yields, a 2.5-fold molar excess of adrenodoxin over the other component was included. In incubations which included cytochrome P-450,, there was also a time-dependent EDC-induced irreversible precipitation of the cytochrome, leading to formation of an apparently severely cross-linked form which did not enter the stacking gel. Inclusion of 10% glycerol reduced, but did not eliminate this precipitation. Comparison of the mobility of adrenodoxin reductase, cytochrome P-450, and their adrenodoxin cross-links by SDSpolyacrylamide gel electrophoresis with standard proteins of known molecular weights (graph not shown) allowed calculation of the apparent molecular weights of these species. Values obtained were52,500 and 50,000 for adrenodoxin reductase and cytochrome P-45OWc,respectively, in good agreement with previous determinations. Both cross-linked species migrated with apparent M, = 63,000, and could not be separated on a variety ofgel systems (6-15% polyacrylamide). Since the molecular weight of adrenodoxin has been reported to be12,000 (26), this migration pattern demonstrates that the cross-linked species in each case is exclusively the 1:l covalently-linked species. Because higher order complexes among adrenodoxin reductase, adrenodoxin, and cytochrome P-450 have beensuggested (12), we carried out cross-linking incubations in the presence of all three components. The photograph (Fig. 4) shows a close-up view of the high molecular weight region beforeand after addition of EDC. Although the adrenodoxin-cytochrome P-450 adrenodoxin-adrenodoxin reductase cross-linked species could not be resolved, it is clear that no higher order (e.g. ternary or quaternary) complex is formed. DISCUSSION For a subclass of electron transport proteins, electrostatic interactions play a role in the functional protein-protein

4 10028 Adrenodoxin Interaction with Reductase and Cytochrome P-450,, X-LINKS AR P450 FIG. 4. Formation of binary cross-linked complexes upon incubation of adrenodoxin reductase, adrenodoxin, and cytochrome P-450,. The photograph shows the high molecular weight region of 6% SDS-polyacrylamide gels of an incubation of all three components 1) before, and 2) 1 h after addition of EDC. Conditions were as described in the legend to Fig. 3,except that in the incubation with all three proteins, 70 p~ adrenodoxin was used. complexes through which electron transfer occurs. In cytochrome c, the heme edgeis exposed to solvent, and theprotein contains a ring of positively charged amino acid residues (eight for horse heart cytochrome c ) which surround the heme edge (27). Cytochrome c peroxidase, a heme protein with which cytochromec forms a 1:l complex, has acomplementary ring of negatively charged residues surrounding its heme, and models indicate that thetwo proteins must interact via charge pairing between the amino and carboxyl groupings(28). These interactions appear to play an important role inthe alignment of the hemes for optimal electron transfer. Chemical modification studies substantiate the importance of such interactions in the interaciton of cytochrome c with cytochrome c oxidase (18), azurin (29), plastocyanin (29), cytochrome b:, (30), and adrenodoxin (20), and in the interaction of cytochrome b5 with cytochrome b5 reductase (19). The finding of protein complexes whose strength of interaction diminishes rapidly with increasing ionic strength furtherimplicates such electrostatic interactions in the adrenodoxin-adrenodoxin reductase (ll),adrenodoxin-cytochrome P-450(14, 31),and ferredoxin reductase-ferredoxin (32) protein pairs. In an earlier publication (20), reaction of adrenodoxin with EDCwasshown to specificallymodify and neutralize the charge of three residues (Glu 74, Asp 79, and Asp 86). Derivatization was shown to diminish the interaction of adrenodoxin with adrenodoxin reductase, based upon K,,, effects in the cytochrome c reductase assay. In the present studies, we haveutilizedtwo different preparations of EDC-modified adrenodoxin, and have shown that the same modifications which lead to diminished binding to NADPH-adrenodoxin reductase also lead to weakened binding to cytochrome P450,,. The extent of the effect of modification upon binding is similar for both interactions. Thus, one or more of the above amino acids appear to be involved inthe interaction of adrenodoxin with both adrenodoxin reductase and cytochrome P-450. The effects of EDC modification of adrenodoxin upon protein complex formation support the idea that a single region of adrenodoxin is involved in the interactions with both the flavoprotein and cytochrome P-450, and thusprovide a phys- ical explanation for the necessity foradrenodoxin to function as a shuttle. While it is likely, based upon their proximity, that all 3 residues participate in both charge pairing interactions, one could also propose the limiting casewherein a residue at one end (e.g. Glu 74) functions exclusively in one protein complex, whilethat atthe other end (Asp 86) participates in the other protein complex, thus allowing a ternary complex among the proteins. Lim and Kimura (33) have analyzed the amino acid sequence of adrenodoxin using the method of Chou and Fasman (34), and have predicted a high degree of a-helix in this region; the derivatized residues are thus likely to be within the same region on the binding surface of adrenodoxin, and binding to the reductase and to the cytochrome should be mutually exclusive. Nevertheless, it is possible that this sequence could exist in a more extended form, and the ternarycomplex modelmight apply. The exclusive binary complex model rather than the ternary complexmodel is substantiated by the EDC cross-linking studies. When adrenodoxin is incubated with either adrenodoxin reductase or cytochrome P-450, in the presence of EDC, a single type of cross-linked species is generated in each case. The migration on SDS-polyacrylamide gels is that expected from a 1:l complex of adrenodoxin plus the other protein. When incubations of all three proteins were carried out in the presence of EDC, nohighermolecularweight complexes were seen; only the apparent M, = 63,000 band was seen, coincident with the disappearance of the 52,000 (adrenodoxin reductase), 50,000 (cytochrome P-450), and 12,000 (adrenodoxin) bands. Thus, from the present studies it appears that when adrenodoxin forms a complex with either the reductase or cytochrome P-450, complex formation with the other partner is precluded; adrenodoxin must therefore bind sequentially to adrenodoxin reductase and tocytochrome P-450 during its electron transfer sequence. Acknowledgment-The expert technical assistance of Linda Vinnedge is gratefully acknowledged. REFERENCES 1. Simpson, E.R., and Boyd, G.S. (1966)Biochem. Biophys. Res. Commun Shikita, M., and Hall, P. F. (1974)Proc. Natl. Acad. Sci. U.S. A. 71, Seybert, D. W., Lancaster, J. R., Jr., Lambeth, J. D., and Kamin, H. (1979)J. Bwl. Chem. 254, Hume, R., and Boyd, G. S. (1978)Biochem. Soc. 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