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H Uhlmann

Publications and source records attributed to H Uhlmann.

15 recordsLinked to original sources
PubMed1998

New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4-108).

BACKGROUND: Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland mitochondrial matrix of mammals. Adx is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where they are consumed in hydroxylation reactions. A crystallographic study of Adx is expected to reveal the structural basis for an important electron transfer reaction mediated by a vertebrate [2Fe-2S] ferredoxin. RESULTS: The crystal structure of a truncated bovine adrenodoxin, Adx(4-108), was determined at 1.85 A resolution and refined to a crystallographic R value of 0.195. The structure was determined using multiple wavelength anomalous dispersion phasing techniques, making use of the iron atoms in the [2Fe-2S] cluster of the protein. The protein displays the compact (alpha + beta) fold typical for [2Fe-2S] ferredoxins. The polypeptide chain is organized into a large core domain and a smaller interaction domain which comprises 35 residues, including all those previously determined to be involved in binding to AR and cytochrome P450. A small interdomain motion is observed as a structural difference between the two independent molecules in the asymmetric unit of the crystal. Charged residues of Adx(4-108) are clustered to yield a strikingly asymmetric electric potential of the protein molecule. CONCLUSIONS: The crystal structure of Adx(4-108) provides the first detailed description of a vertebrate [2Fe-2S] ferredoxin and serves to explain a large body of biochemical studies in terms of a three-dimensional structure. The structure suggests how a change in the redox state of the [2Fe-2S] cluster may be coupled to a domain motion of the protein. It seems likely that the clearly asymmetric charge distribution on the surface of Adx(4-108) and the resulting strong molecular dipole are involved in electrostatic steering of the interactions with AR and cytochrome P450.

Adrenodoxin↗
PubMed1998

Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states.

Site-directed mutants of adrenodoxin were studied for their ability to undergo cluster-iron substitution when reacted with zinc or cadmium salts under non-denaturing conditions in the presence or absence of reductants. Equilibrium and kinetic data for metal substitution were correlated with data on the stability to thermal unfolding and with the redox potential of the protein. Similarly to the wild-type protein, all mutants were able to stabilize a substituted form of the protein containing two metal (Zn or Cd) atoms and two sulfide ions/mol protein and a substituted form of the protein containing two sulfide ions and five Cd atoms/mol protein. However, the distribution of these two metal-substituted forms was different among the investigated proteins. [Ser95]Adrenodoxin stabilized either metal-substituted forms, confirming that Cys95 is not involved in metal coordination, even when five Cd atoms are bound to the protein. Removal of the extremely conserved hydroxy function at position 54 resulted in complete apoprotein formation upon reaction with Cd (75 % with Zn) under reducing conditions, indicating a cluster-harboring role for this function, which is conserved in all known 2Fe-2S proteins. Mutants at His56, which represents a residue unique to most vertebrate-type ferredoxins, were much more reactive than the wild-type protein with either metal, indicating that His56 plays a prominent role in the stabilization of the protein structure in the immediate vicinity of the cluster in this class of proteins. The nature of the metal-substitution products was dependent on cluster accessibility. For the reduced proteins, apoprotein formation depended on protein stability, while the velocity of metal substitution depended on the ease of cluster reduction.

Adrenodoxin↗
PubMed1997

Pro108 is important for folding and stabilization of adrenal ferredoxin, but does not influence the functional properties of the protein.

The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.

Adrenal Glands↗
PubMed1996

Reversible, non-denaturing metal substitution in bovine adrenodoxin and spinach ferredoxin and the different reactivities of [2Fe-2S]-cluster-containing proteins.

The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.

Adrenodoxin↗
PubMed1996

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.

Adrenodoxin↗
PubMed1995

The role of threonine 54 in adrenodoxin for the properties of its iron-sulfur cluster and its electron transfer function.

The amino acid in position 54 of adrenodoxin is strongly conserved among ferredoxins, consisting of a threonine or serine. Its role was studied by analyzing mutants T54S and T54A of bovine adrenodoxin. Absorption, circular dichroism, fluorescence, and electron paramagnetic resonance spectra of mutant T54S show that this substitution has no influence on the formation and stability of the ferredoxin. The redox potential of this mutant, however, was lowered by 55 mV as compared with native adrenodoxin, indicating a role for this residue in redox potential modulation. Incorporation of the iron-sulfur cluster was not impaired in the T54A mutant, although structural features of the oxidized protein were considerably changed. The decreased stability of the T54A mutant as compared with the wild type and mutant T54S indicates that a hydrogen bond donor at this position stabilizes the protein. Both mutants have been shown to be functionally active. Replacement of threonine 54 by serine or alanine, however, leads to rearrangements at the recognition sites for its redox partners. This is reflected by decreased Km and Kd values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased Km value in cytochrome c reduction. Substrate conversion was accelerated (2.2- and 2.4-fold for mutants T54A and T54S, respectively) in the CYP11B1-, but not in the CYP11A1-dependent reaction.

Adrenodoxin↗
PubMed1994

C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450.

The role of the C-terminal region of adrenodoxin was studied by analyzing deletion mutants 4-114 and 4-108 lacking amino acids 1-3 and 115-128 or 109-128, respectively. Absorption spectra of these mutants were found to be identical to that of wild type adrenodoxin. However, EPR and CD studies indicated that the structure of deletion mutants 4-114 and 4-108 differs from that of wild type adrenodoxin. Mutant 4-107, which in addition to residues 109-128 lacks the unique proline 108, showed no EPR spectrum. This indicates that proline 108 plays an essential role for the formation of the iron-sulfur cluster. Deletion of residues 115-128 or 109-128 did not essentially affect adrenodoxin reductase binding as shown by nearly unchanged cytochrome c reduction activity. In a CYP11A1 assay, mutants 4-108 and 4-114 exhibited 3.2- and 5-fold decreased Km values, respectively, whilst the Kd values for CYP11A1 decreased 3- and 1.9-fold, respectively. Additionally, in a CYP11B1 assay, mutants 4-108 and 4-114 showed decreased Km values. Furthermore, the first step of electron transfer to CYP11B1, but not to CYP11A1, was accelerated up to 4.5-fold by the adrenodoxin mutants. The results suggest that the C-terminal peptide of adrenodoxin, especially proline 108, affects the structural integrity of the iron-sulfur cluster and that electron donation from adrenodoxin to CYP11A1 and CYP11B1 is determined at least in part by different features of the cytochromes.

Adrenal Glands↗
PubMed1994

Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli.

Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining. In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied. Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli. By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E. coli, also in the periplasmic space. A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E. coli by antibodies against both proteins.

Adrenal Cortex↗
PubMed1992

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

Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.

Adrenodoxin↗
PubMed1985

[North American blastomycosis].

We report the case of an African patient with North American blastomycosis. The clinical and mycological results and presented and the therapy explained.

Adult↗
PubMed1983

[Scanning electron microscopy studies of the structure of calcium oxalate urinary calculi].

The examination of calcium oxalate calculi under a scanning electron microscope confirmed the 4 structural types distinguished under a polarising microscope using microsections. The fine structure of the radial-concentric-shelled structural type I is characterized as a dense pack of radially ordered prismatic bundles. In combination with energy-dispersive analysis the occurrence of the finely crystalline type II is confirmed. Energy-dispersive analysis shows an unexpected variety of element distribution in the microscopic range.

Calcium Oxalate↗
PubMed1976

[Peritoneoscopy with 2 optical devices: reduced risk - better diagnostic results (author's transl)].

Peritoneoscopy with 2 optical devices was performed in two cases; the technique of this procedure is described. The method allows inspection of areas, which cannot be seen during routine peritoneoscopy, especially of the anterior abdominal wall and of intraabdominal organs covered by adhesions. In addition, this method allows to find out, whether the anterior abdominal wall and vessels in it were injured by the troikart; the risk of the procedure is reduced thereby. Using a second optical device allows to visualize areas, which cannot be studied during routine peritoneoscopy because of intraabdominal adhesions. Thus better diagnostic results can be achieved with this method as compared to the routine technique.

Aged↗
PubMedDate not supplied

Structure-function studies on mutants of adrenal ferredoxin.

Mutants of the adrenal ferredoxin (adrenodoxin) have been expressed in E. coli in order to improve the understanding of its structure and function. Replacement of the ligands to the /2Fe-2S/ center, C46, C52, C55 and C92, by serine, histidine or aspartic acid lead to apoproteins not incorporating the iron-sulfur cluster, whereas C95S forms a functionally active holoprotein. C-terminal deletions up to amino acid 109 affect the conformation around the iron-sulfur cluster in adrenodoxin and the interaction with CYP11A1 and CYP11B1, but not with adrenodoxin reductase. The presence of P108 is necessary for incorporation of the /2Fe-2S/ cluster and obviously for correct folding of adrenodoxin.

Adrenodoxin↗
PubMedDate not supplied

Structure of adrenodoxin and function in mitochondrial steroid hydroxylation.

The three-dimensional structure of a truncated mutant of bovine adrenodoxin has been resolved at 1.85 A resolution by MAD. The protein consists of a large core region and a more flexible hairpin loop bearing residues which have been previously described as being involved in redox partner recognition. To study the role of distinct protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase (AdR), CYP11A1 and CYP11B1, as well as in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis and produced in Escherichia coli, and their structural and functional properties have been characterized in detail. It could be demonstrated that Tyr82 is located at the edge of the flexible interaction loop of adrenodoxin participating in interactions with AdR and P450s. His56, being close to Tyr82, forms a bridge between the core region of adrenodoxin and the interaction loop. Its role in transmitting changes of the cluster region to the interaction site has also been supported by functional studies. Pro108 of adrenodoxin, the only proline residue contained in the protein and being conserved in this position among several other vertebrate-type ferredoxins, has been demonstrated to be of importance for the correct folding of this protein.

Adrenodoxin↗