Adrenodoxin: Structure, stability, and electron transfer properties

Grinberg, Asya V.; Hannemann, Frank; Schiffler, Burkhard; Müller, Jürgen; Heinemann, Udo; Bernhardt, Rita

doi:10.1002/1097-0134(20000901)40:4<590::aid-prot50>3.0.co;2-p

Cited by 195 publications

(194 citation statements)

References 141 publications

Supporting

Mentioning

182

Contrasting

Order By: Relevance

“…The differential effect of these charge alterations at residue T71 on AdR and CYP11A1 association supports the idea that the binding sites on Adx for its interaction partners are overlapping rather than identical, as previously postulated. 22 In the steady-state approximation for the donor-acceptor complex concentration, the pseudo-first-order ET rate is (3) when one reactant is present in excess. 76 In the saturation regime, the quasi-unimolecular rate is k 1 = k ET , denoted k obsd,max in the tables, obtained by fitting the experimental data to eq 3.…”

Section: Discussionmentioning

confidence: 99%

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

et al. 2007

Self Cite

View full text Add to dashboard Cite

Protein phosphorylation is a common regulator of enzyme activity. Chemical modification of a protein surface, including phosphorylation, could alter the function of biological electron-transfer reactions. However, the sensitivity of intermolecular electron-transfer kinetics to post-translational protein modifications has not been widely investigated. We have therefore combined experimental and computational studies to assess the potential role of phosphorylation in electron-transfer reactions. We investigated the steroid hydroxylating system from bovine adrenal glands, which consists of adrenodoxin (Adx), adrenodoxin reductase (AdR), and a cytochrome P450, CYP11A1. We focused on the phosphorylation of Adx at Thr-71, since this residue is located in the acidic interaction domain of Adx, and a recent study has demonstrated that this residue is phosphorylated by casein kinase 2 (CK2) in vitro. 1 Optical biosensor experiments indicate that the presence of this phosphorylation slightly increases the binding affinity of oxidized Adx with CYP11A1 ox but not AdR ox . This tendency was confirmed by K A values extracted from Adx concentration-dependent stopped-flow experiments that characterize the interaction between AdR red and Adx ox or between Adx red and CYP11A1 ox . In addition, acceleration of the electron-transfer kinetics measured with stopped-flow is seen only for the phosphorylated Adx-CYP11A1 reaction. Biphasic reaction kinetics are observed only when Adx is phosphorylated at Thr-71, and the Brownian dynamics (BD) simulations suggest that this phosphorylation may enhance the formation of a secondary Adx-CYP11A1 binding complex that provides an additional electron-transfer pathway with enhanced coupling.

show abstract

Section: Discussionmentioning

confidence: 99%

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

et al. 2007

Self Cite

View full text Add to dashboard Cite

show abstract

“…This Fdx folding motif is among the most abundant in nature, and it has been well studied previously biochemically (18)(19)(20)(21)(22)(23), making Fdx an excellent candidate for quantitative, biophysical characterization. Moreover, the planttype Fdx folding motif has been found in numerous redox proteins and enzymes, as well as in functionally unrelated proteins that do not contain the [2Fe-2S] cluster, such as ubiquitin and the immunoglobulin-binding domain of protein G (24,25).…”

mentioning

confidence: 99%

“…These proteins are key players in the three life-essential processes: photosynthesis, nitrogen fixation, and respiration (29,30). The plant-type Fdx proteins carry a single surface-exposed [2Fe-2S] iron-sulfur cluster (ISC) (19), and homologous proteins are found in all organisms, e.g., adrenodoxin in human mitochondria (20). The oxidation-reduction potentials (E m ) of these Fdx proteins range from −325 mV to around −450 mV (21), making Fdx one of the strongest soluble reducing agents in nature (22).…”

mentioning

confidence: 99%

Allostery in the ferredoxin protein motif does not involve a conformational switch

Nechushtai

Lammert²,

Michaeli³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain planttype ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the ironsulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.electron transfer | functional energy landscape | iron-sulfur proteins | protein folding O ver the last several decades, our understanding of protein function has evolved from a rather static perspective, where signaling and function have been understood through surface complementarity arguments, such as the "lock and key" paradigm (1, 2), to a more dynamic view where protein conformational fluctuations are inextricably linked to function (3). As our understanding of protein dynamics expands, we are revealing many mechanisms by which proteins exploit conformational fluctuations to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations. Because the kinetics of these tra...

show abstract

“…The enzymatic activity of the cytochrome P450-dependent steroid hydroxylases is based on their ability to activate molecular oxygen by reductive splitting of dioxygen. This multistep reaction requires the transfer of electrons from the flavoprotein adrenodoxin reductase (AR) via adrenodoxin (Adx) to the terminal cytochromes P450 as electron acceptors in dependence on the specific hydroxylation substrate (1)(2)(3). Several models for electron transfer have been discussed, including a shuttle model in which Adx forms consecutive 1:1 complexes (4) with AR and cytochrome P450scc and models requiring the formation of an organized 1:1:1 ternary complex (5) or a 1:2:1 quaternary complex (6) between AR, Adx, and cytochrome P450scc.…”

mentioning

confidence: 99%

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

Müller¹,

Лапко²,

Bourenkov³

et al. 2001

Journal of Biological Chemistry

Self Cite

158

122

View full text Add to dashboard Cite

The steroid hydroxylating system of adrenal cortex mitochondria consists of the membrane-attached NADPH-dependent adrenodoxin reductase (AR), the soluble one-electron transport protein adrenodoxin (Adx), and a membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-Å resolution crystal structure of the Adx⅐AR complex, 580 In mitochondria of the adrenal cortex, the cytochrome P450 enzymes of the CYP11 family catalyze the side chain cleavage of cholesterol to form pregnenolone (P450scc, 1 CYP11A1) and are involved in the formation of cortisol (P45011␤, CYP11B1) and aldosterone (P450aldo, CYP11B2) (1). The enzymatic activity of the cytochrome P450-dependent steroid hydroxylases is based on their ability to activate molecular oxygen by reductive splitting of dioxygen. This multistep reaction requires the transfer of electrons from the flavoprotein adrenodoxin reductase (AR) via adrenodoxin (Adx) to the terminal cytochromes P450 as electron acceptors in dependence on the specific hydroxylation substrate (1-3). Several models for electron transfer have been discussed, including a shuttle model in which Adx forms consecutive 1:1 complexes (4) with AR and cytochrome P450scc and models requiring the formation of an organized 1:1:1 ternary complex (5) or a 1:2:1 quaternary complex (6) between AR, Adx, and cytochrome P450scc. Common to these models is a complex between AR and Adx during the first steps of electron transfer from the reductase to the cytochrome P450.Recently, the crystal structures of two forms of bovine adrenodoxin (7, 8) and of adrenodoxin reductase (9) were determined. These structures revealed the general topology of the two proteins and the molecular environments of the [2Fe-2S] cluster of Adx and the FAD moiety of AR. Here, we report the 2.3-Å resolution crystal structure of a cross-linked 1:1 complex of full-length Adx and AR. This structure shows the geometry of an electron transfer complex of soluble, freely dissociable proteins from a higher eukaryote for the first time, highlights structural adaptations that accompany the binding of AR to Adx, and permits us to predict electron transport paths in their complex. EXPERIMENTAL PROCEDURESSample Preparation-Recombinant bovine Adx and AR were purified and crystallized as described (10). The synthesized Adx differs from the wild-type protein by the exchange of Ser 1 for glycine and is composed of 128 amino acids, including the N-and C-terminal residues missing in the truncated adrenodoxin, Adx-(4 -108), studied earlier (7). Crosslinking of AR to Adx has also been described (11,12). The native complex is formed at low ionic strength between the two proteins, and the cross-linking was carried out with the water-soluble coupling reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, purchased from Sigma). The mixture of Adx (1.2 mol) and AR (300 nmol) was dialyzed for 18 -20 h against 20 mM potassium phosphate, pH 7.2, followed by addition of an equal volume of fresh 8 mM EDC solution in distilled water and incubation at 4°C in the dark wi...

show abstract

Adrenodoxin: Structure, stability, and electron transfer properties

Cited by 195 publications

References 141 publications

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

Allostery in the ferredoxin protein motif does not involve a conformational switch

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

Contact Info

Product

Resources

About