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

Лапко, А. Г.; Muller, Alexander J.; Heese, Olaf; Ruckpaul, K; Heinemann, Udo

doi:10.1002/(sici)1097-0134(199706)28:2<289::aid-prot16>3.0.co;2-e

Cited by 13 publications

(7 citation statements)

References 25 publications

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“…Moreover, both Fd types are negative monopoles with a clear charge separation pointing to a region located in between the interaction domain and the [2Fe−2S] cluster. Thus, it is expected that in an initial approach the Adx negative monopole will focus the Adx [2Fe−2S] centre towards the Fd interaction domain of FNR, which is positively charged, as occurs in the physiological FNR/Fd and AdR/Adx interactions [5,18,28,39]. Our data clearly prove that such FNR/Adx interaction is taking place and that it might support ET.…”

Section: Discussionsupporting

confidence: 62%

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

Faro

Schiffler

Heinz

et al. 2003

European Journal of Biochemistry

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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.Keywords: adrenodoxin; electron transfer; ferredoxin-NADP + reductase; protein-protein interaction.Many biological processes depend on protein-protein electron transfer (ET) reactions, where the specific interaction of a reduced protein with its oxidized counterpart is required [1,2]. The fact that many of the proteins involved in these reactions are able to interact with different partners raises the question about the nature of their interaction surfaces. This can be demonstrated by proteins like ferredoxins (Fd), small [2Fe)2S] proteins that are involved in a multitude of reactions in microorganisms, plants and animals. In the case of Anabaena, a photosynthetic nitrogen-fixing cyanobacterium, Fd is involved in the recognition of the photosystem I and also of several enzymes such as ferredoxin-NADP + reductase (FNR), nitrate and nitrite reductase, glutamate synthase or thioredoxin reductase [3]. This suggests that although the overall structures of these proteins differ widely, their Fd interaction surface should contain some common features. Moreover, it is known that in Anabaena, FNR can recognize not only Fd but also flavodoxin (Fld), a small FMN-containing protein that is synthesized under conditons of iron deficiency when it replaces Fd in the ET from photosystem I to FNR [4]. Th...

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Section: Discussionsupporting

confidence: 62%

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

Faro

Schiffler

Heinz

et al. 2003

European Journal of Biochemistry

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“…Preparation and purification of recombinant bovine Adx and AR has been described recently [18,19]. The Adx used for the crosslink experiments differs from the wild‐type protein by the exchange of Ser1 to Gly1, thereby reducing proteolysis.…”

Section: Methodsmentioning

confidence: 99%

“…Crosslinking of AR to Adx has been described previously [18]. The native complex of the two proteins is formed at low ionic strength, and the crosslinking is carried out with the water‐soluble coupling reagent EDC (Sigma, St Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The colored fraction was pooled and applied on a 2.4 × 10 cm DEAE‐Fractogel column and washed with two gradient solutions: 10–50 m m potassium phosphate, pH 7.4, for 3 h; 50–100 m m potassium phosphate, pH 7.4, for 3 h. The peak containing the covalently crosslinked complex of the recombinant Adx and AR was subsequently purified on an Adx−Sepharose column to remove residual AR and on an ADP−Sepharose column to remove unbound Adx. The cytochrome c reductase activity of the Adx–AR complex and its activity in the transformation of cholesterol to pregnenolone by P450scc has been shown recently [18], and the complex has been crystallized for X‐ray structure determination [8,18].…”

Section: Methodsmentioning

confidence: 99%

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Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

Müller

Лапко

Otto

et al. 2001

European Journal of Biochemistry

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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 2P450scc 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 adrenodoxin2P450scc 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.

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“…To elucidate the enzyme mechanism in detail, bovine [4–6]and porcine AdR [7]have been crystallized. Further crystals were obtained from bovine AdR cross‐linked with adrenodoxin [8], the structure of adrenodoxin is known [9]. All AdR crystals were apparently not adequate for detailed analysis.…”

Section: Introductionmentioning

confidence: 99%

Chaperone‐assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli

et al. 1999

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Adrenodoxin reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds. After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system. The purified protein was homogeneous enough for reproducible crystallization. The crystals diffracted X-rays isotropically beyond 1.7 A î resolution permitting a structure analysis.z 1999 Federation of European Biochemical Societies.

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

Cited by 13 publications

References 25 publications

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

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

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

Chaperone‐assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli

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

Cited by 13 publications

References 25 publications

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

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

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

Chaperone‐assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli

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

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