Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.

Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.

Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.

Expanding the view of scholarship: introduction.

The Association of American Medical Colleges' Council of Academic Societies (CAS) has a long-standing interest in scholarship as it relates to research, education, and service, the traditional definition of the activities of medical school. The work of Ernest Boyer and Charles Glassick is highly respected for redefining scholarship and conceiving how scholarship as thus defined can be assessed. Because their ideas have been applied in other areas of the academy but not widely in medical faculties, the CAS Task Force on Scholarship collected a special set of papers on Boyer's four areas of scholarship as applied to medical school, including case studies and the perspective from the university. The four areas of scholarship defined by Boyer and Glassick are the scholarship of discovery, the scholarship of integration, the scholarship of application, and the scholarship of teaching. The scholarship of discovery-research-has for decades been the primary focus for promotion and tenure for medical school faculty, even though the faculty also had major and critical activities in the other areas of scholarship. The CAS hopes that the ideas put forth in this special theme issue will produce a continuing dialogue as faculty and administrators at medical schools reflect on the value of these different forms of scholarship, their application by medical school faculty, and their contributions to the individual missions of each medical school and teaching hospital. In addition, these articles will stimulate continuing discussions that will definite equitable methods for the continued assessment of the scholarly accomplishments of medical school faculty.

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria.

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.

The role of various domains of the iron-sulfur protein in the assembly and activity of the cytochrome bc1 complex of yeast mitochondria.

Assembly studies in vitro of deletion mutants of the iron-sulfur protein into the cytochrome bc1 complex revealed that mutants localized in the extramembranous regions of the protein were not assembled into the complex in contrast to the efficient assembly of mutants in the membrane-spanning region. Charged amino acids located in the extramembranous alpha1-beta4 loop and the alpha1 helix were mutated and expressed in yeast cells lacking the gene for the iron-sulfur protein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 as well as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron-sulfur protein suggesting that these amino acids are required to maintain protein stability. By contrast, no loss of iron-sulfur protein accompanied the 30-50% loss of bc1 complex activity in mutants of three conserved alanine residues, A86, A90, and A92, suggesting that these residues may be involved in the proposed movement of the flexible tether of the iron-sulfur protein during catalysis.

The alpha 1-helix in the extra-membranous domain contributes to the stability of the iron-sulfur protein of the cytochrome bc1 complex.

Previously, we reported that several charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein are required to maintain the stability of the protein and its assembly into the cytochrome bc1 complex. The current study extends this analysis to several amino acids localized in the single alpha-helix present in the extra-membranous domain of the protein. Three charged and two uncharged residues in the alpha1-helix were mutated and used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants V132L and H124L grew at half the rate of the wild type in medium containing glycerol/ethanol, while E125Q grew more slowly than the wild type. The rates of growth of T122A and E128Q were identical to that of the wild-type cells. Activity of the cytochrome bc1 complex was decreased 70, 40, and 80% in mutants H124L, E125Q, and V132L, respectively, while the activity of T122A and E128Q was decreased 30 and 20% relative to the wild type. Western blotting experiments revealed that the content of the iron-sulfur protein was decreased in mutants H124L and V132L; however, no decrease in the content of the iron-sulfur protein was observed in the other mutants. These results suggest that several amino acids located in the alpha1-helix of the protein are important in maintaining the stability and proper assembly of the iron-sulfur protein in the bc1 complex.

The role of charged amino acids in the alpha1-beta4 loop of the iron-sulfur protein of the cytochrome bc1 complex of yeast mitochondria.

Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138-153 might be involved in the assembly of this protein into the cytochrome bc1 complex. To determine which specific residues might be involved in the assembly process, charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type. Activity of the cytochrome bc1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c1 content, determined by immunoblotting, was comparable with that of wild-type cells. However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein. The efficiency of assembly in vitro compared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc1 complex.

Dicyclohexylcarbodiimide inhibits proton pumping in ubiquinol:cytochrome c oxidoreductase of Rhodobacter sphaeroides and binds to aspartate-187 of cytochrome b.

In recent studies we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in ubiquinol:cytochrome c oxidoreductase (cytochrome bc1 complex) from yeast mitochondria where it was bound to aspartate-160 of cytochrome b. In the current study, we report that DCCD and its fluorescent analogue, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]-carbodiimide (NCD-4), inhibit 50-60% proton pumping in the cytochrome bc1 complex of the bacterium Rhodobacter sphaeroides with a 20% inhibition of electron transfer activity. Radioactive DCCD is bound exclusively to cytochrome b at aspartate-187, which is located at the C-terminal region of the CD loop connecting membrane-spanning helices C and D of cytochrome b. Fluorescent studies with NCD-4 revealed that aspartate-187 is located in a mildly hydrophobic pocket in the bc1 complex at a distance of 2-3 A from the surface of the membrane.

Exogenous ubiquinol analogues affect the fluorescence of NCD-4 bound to aspartate-160 of yeast cytochrome b.

Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc1 complex. Preincubation of the bc1 complex with the reduced substrate analogues, DQH2, DBH2, and Q6H2 resulted in 20-40% increase in the fluorescence emission intensity of NCD-4 and a 10-20% increase in the binding of [14C]DCCD to the bc1 complex. By contrast, preincubation with the oxidized analogues DQ. DB, and Q6 resulted in a 20-40% decrease in the fluorescence emission intensity of NCD-4 and a 20-40% decrease in the binding of [14C]DCCD to the bc1 complex. Moreover, addition of the reduced ubiquinols to the bc1 complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc1 complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc1 complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc1 complex had no effect on the binding of NCD-4 to the bc1 complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.

The role of the membrane-spanning and extra-membranous regions of the iron-sulfur protein in its assembly into the cytochrome bc1 complex of yeast mitochondria.

The assembly of six deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated by immunoprecipitation from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the intact cytochrome bc1 complex. After import, the mutant proteins lacking residues 41-55 or 66-78, located at the membrane-spanning region of the protein, and residues 182-196 located at the C-terminus of the protein, were assembled in vitro into the bc1 complex approximately 50% as effectively as the wild type iron-sulfur protein suggesting that these regions of the iron-sulfur protein may not be critical for the assembly. By contrast, only trace amounts of the mutant proteins lacking residues 80-95, 122-135, 138-153 located in the extra-membranous region of the iron-sulfur protein were assembled into the bc1 complex. After import in vitro into mitochondria isolated from a cytochrome b-deficient yeast strain, the mutants lacking residues 41-55 and 182-196 were assembled as efficiently as the wild type; however, the mutants lacking residues 55-66 and 66-78 were assembled less efficiently in the absence of cytochrome b suggesting that the hydrophobic membrane-spanning region, residues 55-78, of the iron-sulfur protein, may interact with cytochrome b during the assembly of the bc1 complex.

Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria.

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc1 complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212-16218]. The deletion mutants lacking amino acid residues 55-66 or residues 161-180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161-180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc1 complex; however, the protein lacking residues 55-66 was assembled in vitro into the bc1 complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55-66. This deletion mutant protein was also assembled into the bc1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55-66, is not required for assembly of the iron-sulfur protein into the bc1 complex; however, this association did not lead to enzymatic activity of the bc1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.

Capturing the promise of science in medical schools.

To gain a better understanding of the effects on medical schools of transformations in medical practice, science, and public expectations, the Association of American Medical Colleges (AAMC) constituted the Advisory Panel on the Mission and Organization of Medical Schools (APMOMS) in 1994. APMOMS created six working groups to address the issues deemed by panel members to be of highest priority. This article is a report of the findings of the Working Group on Capturing the Promise of Medical Research, which addressed questions concerning the direction of research and the integration of scientific developments in medical education and practice. The working group explored a broad panorama of issues, including those related to sustaining the accomplishments, momentum, and progress of medical research. A dominant theme emerged: the central importance of an environment of discovery to the core missions of medical schools. The present article consists of the group's comments and recommendations on the main topic-the promise of biomedical research in relation to medical education-and their comments and recommendations on five other topics that have important relationships to the main topic and to the group's central charge. These are ethics; academia-industry relations; the administrative structure of medical schools; university-medical school relations; and research funding.

The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei.

The mitochondrial electron-transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.

Topographical organization of cytochrome b in the yeast mitochondrial membrane determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.

In previous studies, we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bc1 complex from yeast mitochondria and was bound selectively to cytochrome b. Extensive trypsin digestion of [14C]DCCD-labeled cytochrome b isolated from a cytochrome bc1 complex treated with DCCD yielded a single radiolabeled 7.0 kDa peptide with the N-terminus VTLWNVG, indicating that trypsin cleavage had occurred at arginines-110 and -178. This segment of cytochrome b contains one acidic residue, aspartate-160, localized in amphiphilic, non-membrane-spanning, helix cd. To explore the environment of amphiphilic helix cd, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bc1 complex isolated from yeast mitochondria, a fluorescent compound was formed with a 340 nm excitation peak and a 441 nm emission peak. NCD-4 was selectively bound to cytochrome b and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bc1 complex reconstituted into proteoliposomes. Competition experiments and trypsin digestion of NCD-4-labeled cytochrome b indicated that NCD-4 and DCCD were bound to the same site on cytochrome b. The fluorescence of NCD-4 bound to the cytochrome bc1 complex was quenched equally by CAT-16, an amphiphilic spin-label that intercalates at the membrane surface, and 5-doxylstearic acid, a nitroxide derivative of stearic acid, and to a lesser extent by 7-doxylstearic and 12-doxylstearic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Import of subunit VII of the cytochrome bc1 complex into yeast mitochondrial.

The import of subunit VII, the so-called ubiquinol binding protein of the cytochrome bc1 complex of yeast mitochondria, has been investigated by subcloning the gene for subunit VII into the expression vector pSP64. The precursor to subunit VII synthesized in vitro by a transcription/translation system had the same molecular weight as the mature form of this protein present in mitochondria, confirming earlier reports that subunit VII does not contain a cleavable presequence. The subunit VII precursor was imported efficiently into a protease-resistant compartment of isolated yeast mitochondria in a reaction dependent upon a membrane potential and the presence of ATP both in the mitochondrial matrix and in the extramitochondrial space. After import, the radiolabeled subunit VII was bound to the mitochondrial inner membrane through protein-protein interactions and could not be detected in the matrix fraction or in the intermembrane space. Subunit VII was also imported into mitoplasts, where the protein was associated with the inner membrane facing the matrix side of the membrane. By contrast, after import into mitochondria obtained from yeast cells lacking cytochrome b, subunit VII was localized in the supernatant fraction, suggesting that the presence of cytochrome b may be required for the association of subunit VII with the inner mitochondrial membrane.

Isolation and partial characterization of calcium-activated chloride ion channels from thylakoids.

The goal of the present work was to characterize the thylakoid anion channel. Toward this purpose a new, more sensitive, assay was developed to measure chloride channel activity of solubilized and partially purified thylakoid membrane proteins. In this assay potassium channel function was reduced or eliminated by the use of cesium as the coion of chloride. Under these conditions calcium but not magnesium stimulated anion uptake, suggesting the presence of a calcium-activated anion channel in isolated thylakoid membrane proteins. A polyclonal antibody raised against an anion channel isolated from epithelial membranes immunoreacted with three thylakoid membrane proteins; a doublet in the 62 to 66-kDa range and a single band in the 57 to 59-kDa region suggesting that the thylakoid anion channel may be homologous to the mammalian chloride channel. Furthermore, the cDNA which encodes the epithelial chloride channel hybridized on Northern blots prepared from corn total RNA with three mRNA bands.

Sequences of the iron-sulfur protein precursor necessary for its import and two-step processing in yeast mitochondria.

Gene fusion techniques were used to examine whether the presequence of the iron-sulfur protein contains sufficient information for the import of attached mouse cytosolic enzyme dihydrofolate reductase into isolated yeast mitochondria and its subsequent two-step processing. Genes encoding amino-terminal segments of the iron-sulfur precursor protein including the proposed first presequence (residues 1-21), the complete presequence (residues 1-30), and various lengths of the precursor protein from 31 to 160 residues were fused, in frame, to dihydrofolate reductase. All of the fusion proteins, after synthesis in an in vitro transcription-translation system, were imported into a protease-resistant compartment of mitochondria where a single proteolytic cleavage was observed at the first processing site. The second cleavage, however, was not observed after import of any of the chimeric proteins, suggesting that the second cleavage may be strongly influenced by the presence of the passenger protein. A deletion mutant of the iron-sulfur protein precursor lacking residues 161-180 underwent two proteolytic cleavages similar to those observed for the wild-type iron-sulfur protein after import into mitochondria. These results suggest that the complete sequence of the mature form of the iron-sulfur protein including the amino acid residues involved in binding the iron sulfur clusters is not necessary for the second cleavage to occur. In addition, the hydrophobic sequence present in residues 55-66 of the precursor protein which has been suggested to anchor the iron-sulfur protein to the inner membrane, was not necessary for the import and two-step processing of the protein, since a deletion mutant lacking residues 55-66 was processed correctly.

Biochemical evidence for the orientation of cytochrome b in the yeast mitochondrial membrane in the eight-helix model.

The topographical localization of the N-terminus of cytochrome b in the inner mitochondrial membrane was determined by mild proteolysis of the yeast mitochondrial cytochrome bc1 complex and identification of the proteolytic fragments derived from subunits of the complex with an established orientation in the inner membrane. The cytochrome bc1 complex was incorporated into proteoliposomes which were separated by cytochrome c affinity chromatography into two populations in either the mitochondrial or the submitochondrial orientation. Core protein I which protrudes from the matrix side of the inner membrane was digested by proteinase K only in proteoliposomes with the submitochondrial orientation and not in those with the mitochondrial orientation. By contrast, cytochrome c1 with protrudes from the cytoplasmic side of the inner membrane was digested by proteinase K only in proteoliposomes with the mitochondrial orientation and not in those with the submitochondrial orientation. Cytochrome b was digested by SV8 protease only in proteoliposomes with the mitochondrial orientation to yield two aggregating fragments of 25.6 and 24.5 kDa. These peptides were isolated by preparative gel chromatography and sequenced to establish that the cleavage of cytochrome b by SV8 protease occurred at glutamate residues 59 and 66. These residues are localized in the extramembranous loop between the two hydrophobic membrane-spanning helices A and B and thus face the cytoplasmic side of the inner mitochondrial membrane. These results indicate that the N-terminus of yeast cytochrome b protrudes from the matrix side of the inner membrane consistent with the eight-helix model for the orientation of cytochrome b in the membrane.

Oxidation of NADH by a rotenone and antimycin-sensitive pathway in the mitochondrion of procyclic Trypanosoma brucei brucei.

The pathway of NADH oxidation in the procyclic Trypanosoma brucei brucei was investigated in a crude mitochondrial membrane fraction and in whole cells permeabilized with digitonin. NADH:cytochrome c reductase activity was 75% inhibited by concentrations of antimycin that inhibited 95% succinate:cytochrome c reductase activity suggesting that the major pathway for NADH oxidation in the mitochondria involved the cytochrome bc1 complex of the electron transfer chain. Both NADH:cytochrome c and NADH:ubiquinone reductase activities were inhibited 80-90% by rotenone indicating the presence of a complex I-like NADH dehydrogenase in the mitochondrion of trypanosomes. In whole cells permeabilized with low concentrations of digitonin, the oxidation of malate, proline and glucose (in the presence of salicylhydroxamic acid, the inhibitor of the alternate oxidase) was inhibited 30-50% by rotenone. The presence of an alternative pathway for NADH oxidation involving fumarate reductase was indicated by the observation that malonate, the specific inhibitor of succinate dehydrogenase, inhibited 30-35% the rate of oxygen uptake with malate and glucose as substrates in the digitonin-permeabilized cells. We conclude that in the mitochondrion of the procyclic form of T. brucei, NADH is preferentially oxidized by a rotenone-sensitive NADH:ubiquinone oxidoreductase; however, NADH can also be oxidized to some extent by the enzyme fumarate reductase present in the mitochondrion of T. brucei.

Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.

In a recent study [Wang & Beattie (1992) Biochemistry 31, 8445-8459], we reported that dicyclohexylcarbodiimide (DCCD) was bound to either aspartate-155 or glutamate-166 localized in an amphiphilic, non-membrane-spanning, helix of cytochrome. Moreover, DCCD inhibits proton translocation in a cytochrome bf complex reconstituted into proteoliposomes without significant inhibition of electron transfer, suggesting that the helix containing aspartate-155 and glutamate-166 may play a role in proton movements. In order to explore the environment of this amphiphilic helix, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bf complex isolated from spinach chloroplasts, a fluorescent compound was formed with a 331-nm excitation peak and 440-nm emission peak. NCD-4 was selectively bound to cytochrome b6 and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bf complex reconstituted into proteoliposomes. Exhaustive digestion of the NCD-4-labeled cytochrome b6 with trypsin resulted in the formation of a single 6-kDa fluorescent peptide with similar properties to the peptide labeled with radioactive DCCD. The fluorescence of NCD-4 bound to the cytochrome bf complex reconstituted into proteoliposomes was quenched by CAT-16, an amphiphilic spin label that intercalates at the membrane surface, as well as by nitroxide derivatives of stearic acid in the order 5-doxylstearic acid > 7-doxylstearic acid > 12-doxylstearic acid. At higher concentrations, the hydrophilic membrane-impermeant quenchers, CAT-1 and D-569, also quenched the fluorescence of NCD-4.(ABSTRACT TRUNCATED AT 250 WORDS)