[The German University Medicine Map]. / Landkarte Hochschulmedizin.

The University Medicine Map is a major step towards the realization of more transparency regarding the overall services of the medical schools in research, teaching, and patient care within the German university system. It includes comparative information about all 36 medical schools in Germany for the following areas: legal framework, finance, personnel, medical research, teaching and medical education, as well as patient care. The complete set of data for this map is accessible online under www.landkarte-hochschulmedizin.de and a selection of data is also available in print. Advantages and possible political implications for the higher education sector as well as the public domain are illustrated. Finally, the perspectives for future developments are indicated.

[New licensing regulations for physicians. Main areas of reform and first results of the implementation process]. / Die neue Arztliche Approbationsordnung. Schwerpunkte der Reform und erste Erfahrungen mit der Umsetzung.

Since October 2003 medical education in Germany has been given a new more up-to-date basis, the "New Licensing Regulations for Physicians". They represent a reform of both the structure and the content of medical education making great demands on medical faculties and medical students; e.g. elective courses during the first and second cycles of the curriculum, interdisciplinary courses (so-called 'cross-sectional courses'), practical clinical courses of several weeks in five specialties, and family medicine as an option for choice during the last year are central issues of the reform. The number of state-controlled medical examinations has been reduced from four to two. It is now the responsibility of medical faculties to assess the knowledge, clinical skills and professional attitudes of students with respect to the numerous specialties which are part of medical education. This paper presents the essential innovations and describes the current state of the implementation process. It also points out problems that deserve reconsideration. Although the full implementation of the new licensing regulations is still in progress, it might be said that they have already initiated important changes in medical education in Germany.

Isolation, characterisation and crystallisation of a water-soluble fragment of the Rieske iron-sulfur protein of bovine heart mitochondrial bc1 complex.

A water-soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane-bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was + 306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant alpha-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple-wavelength anomalous-scattering technique is underway.

The molecular basis for the natural resistance of the cytochrome bc1 complex from strobilurin-producing basidiomycetes to center Qp inhibitors.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.

Zinc ions inhibit the QP center of bovine heart mitochondrial bc1 complex by blocking a protonatable group.

Bovine heart bc1 complex is reversibly inhibited by zinc ions with an inhibition constant KI of 10(-7) M at pH > or = 7.0. Binding of zinc is at least a factor of 10 tighter than binding of any other metal ion tested. Essentially complete inhibition of ubihydroquinone:cytochrome c oxidoreductase activity is observed at concentrations of [Zn2+] > 5 microM. Zinc does not affect the Km for the substrates, ubihydroquinone or cytochrome c, but zinc inhibits reduction of the cytochromes by ubihydroquinone through the QP center. A radioactive binding assay using 65Zn revealed one high affinity binding site per bc1 complex with KD < or = 10(-7) M at pH = 7.0 and 3-4 additional low affinity binding sites (KD > 2 x 10(-6) M). Zinc binding does not depend on the redox state of the high potential chain (iron-sulfur protein and cytochrome c1). Zinc binds 3 times tighter to Fe-S-depleted bc1 complex indicating that the zinc binding site is not on the "Rieske" iron-sulfur protein in contrast to a recent report by Lorusso et al. (Lorusso, M., Cocco, T., Sardanella, A.M., Minuto, M., Bonomi, F., and Papa, S. (1991) Eur. J. Biochem. 197, 555-561). Zinc binds to a site which has the same affinity for zinc as for protons. We conclude that the zinc binding site is close to a protonatable group of the bc1 complex with pKa = 7.2 which has not been identified previously. We propose that this group is part of the proton channel at the hydroquinone oxidation center of the bc1 complex.

Cytochrome-c oxidase in developing rat heart. Enzymic properties and amino-terminal sequences suggest identity of the fetal heart and the adult liver isoform.

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol-cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densitometrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223-231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.

Human diseases with defects in oxidative phosphorylation. 1. Decreased amounts of assembled oxidative phosphorylation complexes in mitochondrial encephalomyopathies.

The amount of oxidative phosphorylation enzymes in mitochondrial encephalomyopathy patients has been studied by two-dimensional electrophoresis (blue native PAGE/Tricine-SDS-PAGE). Only 20 mg muscle was required to identify and analyse complexes I, III, IV, and V after Coomassie staining. In most cases reduced amounts of the involved complex(es) correlated well with decreased enzyme activities. The reliability of the method was reflected by the constant mutual ratio of the complexes found in all controls. Deviations from normal ratios were found to be more sensitive indicators for a defect than the absolute quantities, which varied considerably within the control group both in the enzymic and in the electrophoretic analysis. The effect of the mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes on the amount of oxidative phosphorylation complexes was demonstrated for the first time directly on the protein level. In patients without known DNA mutations, specific defects of single complexes were identified. The new technique is a sensitive method for the identification of oxidative phosphorylation defects, complementary to enzymic measurements.

Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated with a novel large-scale purification method.

A novel, large-scale method for the purification of cytochrome-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R.A. (1992) J. Biol. Chem. 267, 22,481-22,485]. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (M(r) 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits. We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane-spanning helix. The purified enzyme had a turnover number of 1500 s-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was also true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome-c oxidase purified with our method.

Effect of ethoxyformic anhydride on the Rieske iron-sulfur protein of bovine heart ubiquinol: cytochrome c oxidoreductase.

Treatment of bovine heart ubiquinol-cytochrome c oxidoreductase (complex III, bc1 complex) with ethoxyformic anhydride (EFA) inhibits electron transfer between cytochromes b and c1 [Yagi et al., Biochemistry 21 (1982) 4777-4782]. This paper shows that EFA alters the EPR lineshape of the Rieske iron-sulfur cluster in complex III and in the isolated Rieske protein without a significant decrease of spin concentration. The effect of EFA on the Rieske iron-sulfur cluster is competitive with that of Qo site inhibitors, such as stigmatellin, and is completely reversed by hydroxylamine. These results are consistent with the possible ethoxyformylation by EFA of histidine ligands of the Rieske iron-sulfur cluster at the non-iron binding imidazole nitrogens.

Analysis of cytochrome-b amino acid residues forming the contact face with the iron-sulfur subunit of ubiquinol:cytochrome-c reductase in Saccharomyces cerevisiae.

Four mutations in the mitochondrial cytochrome b of Saccharomyces cerevisiae have been characterized with respect to catalytic properties, inhibitor resistance and subunit interaction. The respiratory-deficient mutant [G137E]cytochrome b and the pseudo-wild-type revertant [G137E, N256K]cytochrome b were described previously [di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332-3339; di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 15750-15757]. Two new mutants [N256K]cytochrome b and [N256I]cytochrome b were isolated by dissociation of the second-site suppressor from the original target mutation. The mutants [G137E]cytochrome b and [G137E, N256K]cytochrome b exhibited a high resistance against methoxyacrylate inhibitors, whereas the suppressors [N256K]cytochrome b and [N256I]cytochrome b showed only a slight resistance. Remarkably, all mutants exhibited stigmatellin cross-resistance. The electron-transfer activity from the substrate nonylubiquinol to cytochrome c of mitochondrial membranes was diminished in all mutants. The substitution G137-->E decreases Vmax/Km by one order of magnitude, indicating a reduced catalytic efficiency for ubiquinol. The amino acid exchange at position 256 to a positively charged lysine residue or to a hydrophobic isoleucine residue resulted mainly in a diminished specific activity. The iron-sulfur subunit and the 8.5-kDa subunit were detectable in all mutants at normal levels in immunoblots of membrane preparations, indicating proper assembly of the complex. However, after purification, the mutant bc1 complex lacked the iron-sulfur subunit and the 8.5-kDa subunit. In contrast, the iron-sulfur subunit can only be dissociated from the parental bc1 complex by harsh treatment. These data suggest that residues 137 and 256 in cytochrome b are crucial for cytochrome-b/iron-sulfur protein interaction.

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.

Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: cytochrome c oxidoreductase?

The Q cycle mechanism of the bc1 complex requires two quinone reaction centers, the hydroquinone oxidation (QP) and the quinone reduction (QN) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of QP site inhibitors to the bc1 complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutant bc1 complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochrome b and parts of the iron-sulfur protein. At least two different binding sites for QP site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochrome b, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the QP pocket is proposed.

Interactions of phospholipids with the mitochondrial cytochrome-c reductase studied by spin-label ESR and NMR spectroscopy.

Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone:cytochrome-c oxidoreductase (bc1 complex) were studied by spin-label electron-spin resonance and by 31P-NMR spectroscopy. Spin-labelled phospholipids were employed to probe the relative binding affinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin-labelled cardiolipin (1,3-bisphosphatidyl-sn-glycerol) and with the spin-labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron-transport activity [Schägger, H., Hagen, T., Roth, B., Brandt, U., Link, T. A. & von Jagow, G. (1990) Eur. J. Biochem. 190, 123-130]. A simplified distribution model showed that neutral phospholipids have much lower protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin-label electron-spin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex. Considerable line narrowing was observed after phospholipase A2 digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.