Mechanism of proton-pumping in the cytochrome b/f complex

Several models have been proposed to interpret the mechanism of proton-pumping associated with the electron transfer reactions in the cytochrome b/f complex. Energetics considerations suggest that the proton pump is coupled to the oxidation of cytochrome b by plastoquinone. Experiments performed in living cells under anaerobic conditions suggest that proton-pumping can occur through two independent mechanisms. When the two b cytochromes are reduced prior to a flash illumination i.e. after a long dark anaerobic incubation (>10 minutes), proton-pumping is very likely associated with the reduction of a semiquinone by cyt b which occurs at a site close to the inner face of the membrane. The electrogenic phase is associated with the tranfer of protons via a transmembrane channel. This process is not inhibited by 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). Under repetitive-flash or under aerobic conditions, proton-pumping occurs according to a modified Q-cycle mechanism, which is inhibited by NQNO.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone

Gaeumannomyces tritici, an ascomycete soilborne fungus, causes a devastating root disease in wheat. Carabrone, a botanical bicyclic sesquiterpenic lactone, is a promising fungicidal agent that can effectively control G. tritici. However, the mechanism of action of carabrone against G. tritici remains largely unclear. Here, we used immunogold for subcellular localization of carabrone and the results showed that carabrone is subcellularly localized in the mitochondria of G. tritici. We then explored the functional analysis of genes GtCytc₁, GtCytb, and GtIsp of the mitochondrial respiratory chain cytochrome bc₁ complex in G. tritici by RNA silencing as a possible target of carabrone. The results showed that the silenced mutant ∆GtIsp is less sensitive to carabrone compared to ∆GtCytc₁ and ∆GtCytb. Compared with the control, the activities of complex III in all the strains, except ∆GtIsp and carabrone-resistant isolate 24-HN-1, were significantly decreased following treatment with carabrone at EC₂₀ and EC₈₀ in vitro (40%–50% and 70%–80%, respectively). The activities of mitochondrial respiratory chain complex III and the mitochondrial respiration oxygen consumption rates in all the strains, except ∆GtIsp and 24-HN-1, were higher with respect to the control when treated with carabrone at EC₂₀ in vivo. The rates of mitochondrial respiration of all strains, except ∆GtIsp, were significantly inhibited following treatment with carabrone at EC₈₀ (ranging from 57% to 81%). This study reveals that the targeting of the iron–sulphur protein encoded by GtIsp is highly sensitive to carabrone and provides a direction for the research of carabrone's target.     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.     

Mitochondrial cytochrome b DNA sequence variation of Atlantic cod from Iceland and Greenland

Sequence variation of a 250–bp (base pair) fragment of the mitochondrial cytochrome b gene has been studied using polymerase chain reaction and direct sequencing of 519 Atlantic cod Gadus morhua from Iceland and 78 cod from Greenland. Twenty-four variable nucleotide sites, mostly silent, define 34 haplotypes. The amount of variation is high ( ĥC =0·73, π=0·52 per 100 bp) with five haplotypes at polymorphic frequencies in Iceland and a number of widely dispersed rather rare haplotypes. A tree of genetic relationships among haplotypes has considerable homoplasy yet it is relatively shallow implying a high turnover of variants of the polymorphism. Net nucleotide genetic divergences among localities are nil. Geographic locality overall area, and inshore/offshore comparison explain none of the variation in an AMOVA, all the variation is among individuals and a null hypothesis of non-differentiation of haplotype frequencies among localities or overall areas cannot be rejected. A temporal year-class effect is found. The evolutionary difference between Greenland and Iceland cod is not significant and the percentage of variation accounted for by the Greenland/Iceland difference is half of what a temporal effect within Iceland explains. There is no evidence for considering the cod at Greenland and Iceland to consist of separate evolutionary units and the question of separate management units must address the lack of diagnostic genotypes and evidence for gene flow from clinal variation.     

Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high‐throughput analyses.     

Phylogenetic relationships of Moxostoma and Scartomyzon (Catostomidae) based on mitochondrial cytochrome b sequence data

A recent phylogenetic study based on morphological, biochemical and early life history characters resurrected the genus Scartomyzon (jumprock suckers, c . eight−10 species) from Moxostoma (redhorse suckers, c . 10–11 species) and advanced the understanding of relationships among species in these two genera, and the genealogical affinities of these genera with other evolutionary lineages within the tribe Moxostomatini in the subfamily Catostominae. To further examine phylogenetic relationships among moxostomatin suckers, the complete mitochondrial (mt) cytochrome b gene was sequenced from all species within this tribe and representative outgroup taxa from the Catostomini and other catostomid subfamilies. Phylogenetic analysis of gene sequences yielded two monophyletic clades within Catostominae: Catostomus + Deltistes + Xyrauchen + Erimyzon + Minytrema and Moxostoma + Scartomyzon + Hypentelium + Thoburnia . Within the Moxostomatini, Thoburnia was either unresolved or polyphyletic; Thoburnia atripinnis was sister to a monophyletic Hypentelium . In turn, this clade was sister to a monophyletic clade containing Scartomyzon and Moxostoma . Scartomyzon was never resolved as monophyletic, but was always recovered as a polyphyletic group embedded within Moxostoma , rendering the latter genus paraphyletic if ' Scartomyzon ' continues to be recognized. Relationships among lineages within the Moxostoma and' Scartomyzon 'clade were resolved as a polytomy. To better reflect phylogenetic relationships resolved in this analysis, the following changes to the classification of the tribe Moxostomatini are proposed: subsumption of' Scartomyzon 'into Moxostoma ; restriction of the tribe Moxostomatini to Moxostoma ; resurrect the tribe Erimyzonini, containing Erimyzon and Minytrema , classified as incertae sedis within Catostominae; retain the tribe Thoburniini.     

Functional activities of monomeric and dimeric forms of the chloroplast cytochrome b6f complex

A monomeric form of the isolated cytochrome b6f complex from spinach chloroplast membranes has been isolated after treatment of the dimeric complex with varying concentrations of Triton X-100. The two forms of the complex are similar as regards electron transfer components and subunit composition. In contrast to a previous report (Huang et al. (1994) Biochemistry 33: 4401–4409) both the monomer and dimer are enzymatically active. However, after incorporation of the respective complexes into phospholipid vesicles, only the dimeric form of the cytochrome complex shows uncoupler sensitive electron transport, an indication of coupling of electron transport to proton translocation. The absence of this activity with the monomeric form of the cytochrome complex may be related to an inhibition by added lipids.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - mega-9- nonanoyl-N-methylglucamide     

Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA

The complete cytochrome b and the control region of mtDNA (about 2070 bp in total) of 10 strains belonging to three subspecies of the common carp, including three wild subspecies (the Yangtze River wild common carp – Cyprinus carpio haematopterus , Yuanjiang River wild common carp – Cyprinus carpio rubrofuscus and Volga River wild common carp – Cyprinus carpio carpio ) and seven domestic strains (Xingguo red carp, Russian scattered scaled mirror carp, Qingtian carp, Japanese Koi carp, purse red carp, Big-belly carp, German mirror carp) were sequenced. Phylogenetic analysis indicated that the 10 strains form three distinct clades, corresponding to C. c. haematopterus , C. c. rubrofuscus and C. c. carpio respectively. Purse red carp, an endemic domestic strain in Jiangxi province of China, showed a higher evolution rate in comparison with the other strains of C. c. haematopterus , most probably because of intensive selection and a long history of domestication. Base variation ratios among the three subspecies varied from 0.78% (between C. c. haematopterus and C. c. rubrofuscus ) to 1.47%(between C. c. carpio and C. c. rubrofuscus ). The topography of the phylogenetic tree and the geographic distribution of three subspecies closely resemble each other. The divergence time between C. c. carpio and the other two subspecies was estimated to be about 0.9 Myr and about 0.5 Myr between C. c. haematopterus and C. c. rubrofuscus . Based on phylogenetic analysis, C. c. rubrofuscus might have diverged from C. c. haematopterus .     

The mechanism of coupling between oxido‐reduction and proton translocation in respiratory chain enzymes

The respiratory chain of mitochondria and bacteria is made up of a set of membrane‐associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane‐associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co‐operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X‐ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone‐cytochrome c oxidoreductase and NADH‐ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.     

Co‐amplification of cytochrome b and D‐loop mtDNA fragments for the identification of degraded DNA samples

We propose a simple and effective approach to simultaneously co‐amplify both cytochrome b and D‐loop fragments to evaluate DNA preservation and to monitor possible contaminations in the analysis of degraded animal DNA samples. We have applied this approach to over 200 ancient salmon samples and 25 ancient whale DNA samples, clearly demonstrating its multiple benefits for analysis of degraded DNA samples, and the ease in which co‐amplification can be optimized for different taxa. This simple, cost‐efficient and genomic DNA‐saving approach can be used routinely in the analysis of minute and degraded DNA samples in wildlife forensics, food inspection, conservation biology and ancient faunal remains.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.     

Further studies on the binding of DCCD to cytochromeB and subunit VIII of complex III isolated from beef heart mitochondria

Complex III (the cytochromeb-c ₁ complex) from beef heart mitochondria was incubated with [¹⁴C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochromeb) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [¹⁴C]DCCD resulted in a 50% decrease in the binding of DCCD to both chtochromeb and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochromeb and subunit VIII of complex III from beef heart mitochondria and suggest that cytochromeb may play a role in proton translocation.     

Mutations of cytochrome b6 in Chlamydomonas reinhardtii disclose the functional significance for a proline to leucine conversion by petB editing in maize and tobacco

We have introduced a proline codon in place of a leucine codon at position 204 of the petB gene of Chlamydomonas reinhardtii. This gene modification mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observed no editing at this position in C. reinhardtii, independent of the type of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototrophic and displayed a block in photosynthetic electron transfer, consistent with a lack of cytochrome b6f activity. Thus the presence of a proline residue at position 204 in cytochrome b6 is detrimental to photosynthesis. We show that the mutant phenotype arose from a defective assembly of cytochrome b6f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical comparison of the proline-containing transformants with a cytochrome b6 mutant deficient in heme-attachment indicates that their primary defect is at the level of assembly of apocytochrome b6 with the bh heme, thereby preventing assembly of the whole cytochrome b6f complex.     

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na⁺/H⁺ antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH 7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.     

Mitochondrial respiratory chain disorders in childhood: Insights into diagnosis and management in the new era of genomic medicine

Background

Mitochondrial respiratory chain disorders (MRCDs) are some of the most common metabolic disorders presenting in childhood, however because of it clinical heterogeneity, diagnosis is often challenging. Being a multisystemic disorder with variable and non-specific presentations, definitive diagnosis requires a combination of investigative approaches, and is often a laborious process.

Scope of review

In this review we provide a broad overview of the clinical presentations of MRCDs in childhood, evaluating the different diagnostic approaches and treatment options, and highlighting the recent research advances in this area.

Major conclusions

Extensive research over the years has significantly increased the frequency with which accurate diagnosis is being made, including the identification of new biomarkers and next generation sequencing (NGS) technologies. NGS has provided a breakthrough in unravelling the genetic basis of MRCDs, especially considering the complexity of mitochondrial genetics with its dual genetic contributions.

General significance

With an increased understanding of the pathophysiology of this group of disorders, clinical trials are now being established using a number of different therapeutic approaches, with the hope of changing the focus of treatment from being largely supportive to potentially having a positive effect on the natural history of the disorder.This article is part of a Special Issue entitled: Special Issue: Frontiers of Mitochondria IG000218.     

Genetic variability of respiratory complex abundance,organization and activity in mouse brain

Mitochondrial dysfunction is implicated in the etiology and pathogenesis of numerous human disorders involving tissues with high energy demand. Murine models are widely used to elucidate genetic determinants of phenotypes relevant to human disease, with recent studies of C57BL/6J (B6), DBA/2J (D2) and B6xD2 populations implicating naturally occurring genetic variation in mitochondrial function/dysfunction. Using blue native polyacrylamide gel electrophoresis, immunoblots and in‐gel activity analyses of complexes I, II, III, IV and V, our studies are the first to assess abundance, organization and catalytic activity of mitochondrial respiratory complexes and supercomplexes in mouse brain. Remarkable strain differences in supercomplex assembly and associated activity are evident, without differences in individual complexes I, II, III or IV. Supercomplexes I₁III₂IV_2–3 exhibit robust complex III immunoreactivity and activities of complexes I and IV in D2, but with little detected in B6 for I₁III₂IV₂, and I₁III₂IV₃ is not detected in B6. I₁III₂IV₁ and I₁III₂ are abundant and catalytically active in both strains, but significantly more so in B6. Furthermore, while supercomplex III₂IV₁ is abundant in D2, none is detected in B6. In aggregate, these results indicate a shift toward more highly assembled supercomplexes in D2. Respiratory supercomplexes are thought to increase electron flow efficiency and individual complex stability, and to reduce electron leak and generation of reactive oxygen species. Our results provide a framework to begin assessing the role of respiratory complex suprastructure in genetic vulnerability and treatment for a wide variety of mitochondrial‐related disorders .     

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1^?1) were added. Results indicated that ascorbate at 100 µmol 1^?1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1^?1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1^?1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright © 2010 John Wiley & Sons, Ltd.