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

Burova, Tatiana V.; Bernhardt, Rita; Pfeil, Wolfgang

doi:10.1002/pro.5560040510

Cited by 23 publications

(1 citation statement)

References 33 publications

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“…Exergonic reactions observed by DSC are usually associated with protein aggregates,32 but in this case no precipitation is observed. Exothermic transitions have been observed also during DSC studies on bovine adrenodoxin, a [2Fe2S] containing protein, and have been suggested to correspond to the decomposition of the Fe–S center 33. We have thoroughly investigated this possibility in Aa Fd using visible spectroscopy and colorimetric quantitation of released iron using the chelator bathophenantroline‐disulphonate (BPS).…”

Section: Resultsmentioning

confidence: 98%

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly

Leal

Gomes

2007

Proteins

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The biological insertion of iron-sulfur clusters (Fe-S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of nonnative protein conformations in iron-sulfur proteins is relevant for the understanding of the folding process and cofactor assembly. We have investigated the formation of a molten globule state in the [3Fe4S][4Fe4S] ferredoxin from the thermophilic archaeon Acidianus ambivalens (AaFd), which also contains a structural zinc site. Biophysical studies have shown that, at acidic pH, AaFd retains structural folding and metal centers. However, upon increasing the temperature, a series of successive modifications occur within the protein structure: Fe-S disassembly, loss of tertiary contacts and dissociation of the Zn(2+) site, which is simultaneous to alterations on the secondary structure. Upon cooling, an apo-ferredoxin state is obtained, with characteristics of a molten globule: compactness identical to the native form; similar secondary structure evidenced by far-UV CD; no near-UV CD detected tertiary contacts; and an exposure of the hydrophobic surface evidenced by 1-anilino naphthalene-8-sulfonic acid (ANS) binding. In contrast to the native form, this apo ferredoxin state undergoes reversible thermal and chemical unfolding. Its conformational stability was investigated by guanidinium chloride denaturation and this state is approximately 1.5 kcal mol(-1) destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe-S pocket probed the conformational dynamics of the molten globule state: fluorescence quenching, red edge emission shift analysis and resonance energy transfer to bound ANS evidenced a restricted mobility and confinement within a hydrophobic environment. The possible physiological relevance of molten globule states in Fe-S proteins and the hypothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed.

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Section: Resultsmentioning

confidence: 98%

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly

Leal

Gomes

2007

Proteins

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Adrenodoxin: Structure, stability, and electron transfer properties

et al. 2000

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Adrenodoxin is an iron‐sulfur protein that belongs to the broad family of the [2Fe‐2S]‐type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH‐dependent adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate‐type ferredoxin, the truncated adrenodoxin Adx(4‐108), that offers the unique opportunity for better understanding of the structure‐function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe‐2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein‐protein recognition and kinetics of adrenodoxin dependent reactions. Proteins 2000;40:590–612. © 2000 Wiley‐Liss, Inc.

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The role of a conserved tyrosine residue in high‐potential iron sulfur proteins

et al. 1995

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Conserved tyrosine-12 of Ectofhiorhodospira hdophikz high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y 12H), tryptophan (Y 12W), isoleucine (Y 12I), and alanine (Y 12A). Variants Y 12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y 12F, Y12H, and Y 12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-1 1 .O. Characterization of the Y 12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 0 6 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-591 Val-58 peptide bond, thereby perturbing Gly-46. The AAG;j.. of Y12F of 2.3 kcal/mol with respect to rcWT HiPlP (25 "C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y 12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 "C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

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

Cited by 23 publications

References 33 publications

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly

Adrenodoxin: Structure, stability, and electron transfer properties

The role of a conserved tyrosine residue in high‐potential iron sulfur proteins

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