Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 FE2S/ cluster ligands

Uhlmann, Heike; Beckert, Vita; Schwarz, Dieter; Bernhardt, Rita

doi:10.1016/0006-291x(92)91349-u

Cited by 109 publications

(88 citation statements)

References 13 publications

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“…The observed ability of Adx to unfold reversibly without dissociation of the iron-sulfur cluster raises the question of whether the holo or the apo protein is involved in in vivo processes such as intracellular transport and folding. The observation that functionally active Adx can be expressed in the periplasm of E. coli (Uhlmann et al, 1992) supports the possibility of transport of the holo protein from the cytoplasm, where the iron-sulfur cluster can be assembled under reductive conditions, to the periplasm.…”

Section: ~~mentioning

confidence: 56%

“…It can be argued that mercaptoethanol prevents an interchange of cysteine residues. Adx possesses five cysteine residues, four of them are involved in the formation of the iron-sulfur cluster (Cys-46, -52, -55, and -92) (Uhlmann et al, 1992). The problem remains to be solved by studying the stability of mutant proteins in which Cys-95 is replaced.…”

Section: ~~mentioning

confidence: 99%

“…Adx was obtained as described by Uhlmann et al (1992). The protein was functionally active with the cytochromes c, P45011 A 1, and P45011B1 as electron acceptors.…”

Section: Adx Preparationmentioning

confidence: 99%

“…The Adx samples were homogeneous as judged by mass spectrometry, electrophoresis, HPLC, and amino acid analysis (Uhlmann et al, 1994). The ratio of optical density at 415 and 276 nm was Q 2 0.92 (Uhlmann et al, 1992). Adx was used immediately after its preparation.…”

Section: Adx Preparationmentioning

confidence: 99%

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Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study

1995

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Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size-exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron-sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at T,, = 46-57 "C, depending on the Na2S concentration with a denaturation enthalpy of A H = 300-380 kJ/mol. From AH versus T,, a heat capacity change was determined as AC, = 7.5 * 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy ( A H = 93 14 kJ/mol at T, = 37.4 k 3.3 "C) and the higher proteolytic susceptibility. The importance of the iron-sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed.

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Section: ~~mentioning

confidence: 56%

Section: ~~mentioning

confidence: 99%

“…Adx was obtained as described by Uhlmann et al (1992). The protein was functionally active with the cytochromes c, P45011 A 1, and P45011B1 as electron acceptors.…”

Section: Adx Preparationmentioning

confidence: 99%

Section: Adx Preparationmentioning

confidence: 99%

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Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study

1995

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“…Protein Purification-Recombinant Adx and AdR were purified as described (21,22). Protein concentration was calculated using ⑀ 414 ϭ 9.8 (mM cm) Ϫ1 for Adx (23) and ⑀ 450 ϭ 11.3 (mM cm) Ϫ1 for AdR (24).…”

Section: Methodsmentioning

confidence: 99%

The Loop Region Covering the Iron-Sulfur Cluster in Bovine Adrenodoxin Comprises a New Interaction Site for Redox Partners

Hannemann¹,

Rottmann²,

Schiffler³

et al. 2001

Journal of Biological Chemistry

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The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as hydroxyl function. A corresponding residue is missing in the cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49⌬, were generated and expressed in Escherichia coli. CD spectra of purified proteins indicate changes of the [2Fe-2S] center geometry only for mutant T49⌬, whereas NMR studies reveal no transduction of structural changes to the interaction domain. The redox potential of T49⌬ (؊370 mV) is lowered by ϳ100 mV compared with wild type adrenodoxin and reaches the potential range of planttype ferredoxins (؊305 to ؊455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49⌬ to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced. These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1. In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1.[2Fe-2S] ferredoxins are found in all organisms from archaea and bacteria to higher plants and animals and function as mediators of electron transfer in a range of multicomponent redox systems (1). The redox active prosthetic group in this class of ferredoxins is characterized by an iron-sulfur cluster, consisting of two non-heme iron ions ligated to thiolate side chains of four cysteines of the polypeptide, bridged by two inorganic sulfide ions.[2Fe-2S] ferredoxins are classified by structure and function into plant-type ferredoxins and vertebrate-type ferredoxins. Vertebrate-type [2Fe-2S] ferredoxins, present in oxygenase systems of bacteria and vertebrates, transfer electrons from a NAD(P)H-dependent ferredoxin reductase to different cytochrome P450 enzymes (2). In vertebrates, ferredoxins of the [2Fe-2S] protein family are present in adrenal cortex, placenta, liver, kidney, and brain (3), where they participate in cytochrome P450-catalyzed hydroxylation reactions to produce steroid hormones, vitamin D metabolites, and bile acids.Bovine Adx 1 as a member of vertebrate [2Fe-2S] ferredoxins, functions as electron mediator from the isoalloxazin system of adrenodoxin reductase (AdR) to the heme iron of two cytochromes P450, CYP11A1 and CYP11B1, localized in the inner mitochondrial membrane of the adrenal cortex (4). CYP11A1 converts cholesterol to pregnenolone, and CYP11B1 catalyzes the 11␤-hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol and the production of aldosterone.The crystal structures of a truncated bovine Adx (5) and of a full-length Adx (6) determined at 1.85-and 2.5-Å resolution, r...

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

et al. 1997

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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 P6(1)22 or P6(5)22, with a = 93.26 A and c = 612.20 A and diffract to 2.9 A resolution at 100 K. Assuming two cross-linked complexes per asymmetric unit yields a reasonable V(M) of 2.97 A3/Da.

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Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 FE2S/ cluster ligands

Cited by 109 publications

References 13 publications

Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study

Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study

The Loop Region Covering the Iron-Sulfur Cluster in Bovine Adrenodoxin Comprises a New Interaction Site for Redox Partners

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

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