Comparative study of bovine adrenodoxin and renorenodoxin

The ferredoxin from bovine renal mitochondria (renoredoxin) has been obtained in a highly purified state. The A415/A280 ratio of the purified renoredoxin is 0.84. The absorption spectrum of renoredoxin was shown to be identical to that of bovine adrenodoxin. Two forms of renoredoxin (Mr 14200 and 13300) were detected by using polyacrylamide gel electrophoresis. These forms exhibit a very similar immunologic cross-reactivity with polyclonal antibodies to adrenodoxin. The N-terminal amino acid sequence of renal ferredoxin was shown to be identical to that of adrenodoxin; the C-terminal sequences of both ferredoxins undergo a similar post-translational proteolytic modification. The amino acid composition of ferredoxins are also very close. Renal ferredoxin can be replaced by adrenodoxin in reconstituted systems from bovine adrenal cortex mitochondria which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.     

Crystallization and X-ray examination of bovine adrenodoxin.

Crystals of adrenodoxin from bovine adrenocortical mitochondria were obtained by the hanging-drop vapor diffusion technique. The crystals belong to a hexagonal crystal lattice with cell parameters 172.50 A and 183.49 A. There are 12 molecules in the asymmetric unit. The crystals diffract to beyond 4.0 A resolution.     

Preparation and crystallization of a cross-linked complex of bovine adrenodoxin and adrenodoxin reductase

Bovine adrenodoxin was cross-linked to adrenodoxin reductase with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Mass spectrometry showed the reaction product to be a 1:1 complex of the two proteins with M_r = 64,790 ± 50. The cross-linked complex showed cytochrome c reductase activity and could be crystallized by hanging-drop vapor diffusion. Crystals of the adrenodoxin-adrenodoxin reductase complex are hexagonal, space group P6₁22 or P6₅22, with a = 93.26 Å and c= 612.20 Å and diffract to 2.9 Å resolution at 100 K. Assuming two cross-linked complexes per asymmetric unit yields a reasonable V_M of 2.97 ų/Da. Proteins 28:289–292, 1997. © 1997 Wiley-Liss Inc.     

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the tac promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.     

Structural organization of adrenodoxin using limited proteolysis

The microheterogeneity of adrenodoxin preparation was established by endogenous proteolysis. The controlled limited trypsinolysis and endogenous proteolysis result in modification of the COOH-terminus of the polypeptide chain with a formation of a protein with Mr = 10 000. The interaction of this protein and of the native protein with cholesterol-specific cytochrome P-450 and adrenodoxin reductase occurs in a similar way.     

Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae

Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.     

Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.     

The tertiary structure of full-length bovine adrenodoxin suggests functional dimers

The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 A resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2(1) with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 A, b = 77.03 A, c = 59.68 A, and beta = 94.83 degrees. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4-108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.     

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc. Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system.

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.     

Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex

cDNA clones for bovine adrenodoxin reductase were isolated, and the primary structure of the enzyme precursor was deduced from their nucleotide sequences. The precursor consists of 492 amino acids including an extrapeptide of 32 amino acids at the amino terminus. The extrapeptide is hydrophilic [corrected] and rich in arginine. The amino terminal sequence of the precursor is homologous with that of the adrenodoxin precursor. A possible FAD- or NADPH-binding site is present near the amino terminus of the mature enzyme.     

Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state

Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.