Respiratory Competence Research Articles

Dose response effect of cement dust on respiratory muscles competence in cement mill workers

Electromyography (EMG) of respiratory muscles is a reliable method of assessing the ventilatory muscle function, but still its use has not been fully utilized to determine the occupational and environmental hazards on respiratory muscles. Therefore, EMG of intercostal muscles was performed to determine the dose response effect of cement dust on respiratory muscles competence. Matched cross-sectional study of EMG in 50 non-smoking cement mill workers with an age range of 20 – 60 years, who worked without the benefit of cement dust control ventilation or respiratory protective devices. EMG was performed by using surface electrodes and chart recorder. Significant reduction was observed in number of peaks (p < 0.0005), maximum peak amplitude (p < 0.0005), peak-to-peak amplitude (p < 0.0005) and duration of response (p < 0.0005) in cement mill workers compared to their matched control. Cement dust impairs the intercostal muscle competence and stratification of results shows a dose-effect of years of exposure in cement mill.

Leukemia Associated Mutants of SHP2 Protein Tyrosine Phosphatase Prevent HoxA10 Tyrosine Phosphorylation in Differentiating Myeloid Cells, Thereby Blocking Transcription of the Gene Encoding Gp91phox.

Gp91phox is a component of the membrane bound, catalytic unit of the phagocyte respiratory burst oxidase. Transcription of the CYBB gene, which encodes gp91phox, is restricted to phagocytes that have differentiated beyond the promyelocyte stage. Transcription continues in mature myeloid cells until cell death and is increased by inflammatory mediators such as LPS or interferon gamma as part of the system of host defense. Previously, we determined that a negative cis element in the CYBB promoter binds a multi-protein complex in undifferentiated myeloid cells which includes the homeodomain proteins HoxA10 and Pbx1, and the histone deacetylase HDAC2. During cytokine induced differentiation, binding of this complex decreases, permitting binding of positive trans factors which activate an adjacent cis element in the CYBB promoter. In previous studies, we also determined that tyrosine phosphorylation decreases HoxA10 binding affinity for the CYBB repressor element. We found that HoxA10 is tyrosine phosphorylated in response to treatment of myeloid progenitor cells and myeloid cell lines with hematopoietic cytokines such as G-CSF, M-CSF and interferon gamma. In undifferentiated myeloid cells, HoxA10 is maintained in a non-tyrosine phosphorylated state by the protein tyrosine phosphatases SHP1 and SHP2. We find that activity of these PTPs decreases during myeloid differentiation. This decrease in PTP activity contributes to increased HoxA10 tyrosine phosphorylation, resulting in CYBB gene transcription as differentiation proceeds. Recently, leukemia associated, activating mutations of SHP2-PTP have been described in human subjects with Juvenile Chronic Myelomonocytic Leukemia (JMMoL), AML and MDS. These SHP2 mutations induce a conformational change which unmasks the PTP domain, resulting in constitutive activation. In these studies, we find that expression of one such leukemia associated SHP2-mutant impairs HoxA10 tyrosine phosphorylation during myelopoiesis. This results in sustained repression of CYBB transcription and decreased gp91phox expression in differentiating cells. Since gp91phox is one of the rate limiting oxidase component proteins during myeloid differentiation and the inflammatory response, activating mutants of SHP2-PTP would therefore be expected to impair respiratory burst competence in malignant myeloid cells. This could contribute to immuno-deficiency in some forms of myeloid malignancy where differentiation appears histologically preserved, such as JMMoL or MDS. Further investigation of the role of SHP2-mutation in phagocyte functional competence may identify rationale molecular targets to ameliorate immuno-deficiency in chronic myeloid diseases.

Investigating the Qn Site of the Cytochrome bc1 Complex in Saccharomyces cerevisiae with Mutants Resistant to Ilicicolin H, a Novel Qn Site Inhibitor

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.

Scaling Methods of Sediment Bioremediation Processes and Applications

AbstractBioremediation has been argued to be one of the most cost‐effective remediation technologies available to reduce soil, sediment, or groundwater contamination, particularly because this approach may allow for the implementation of in‐place strategies. Recent trends have advocated the application of innovative sediment stabilization strategies through placement of (reactive) capping material to allow long‐term biodegradation of contaminants in these complex biogeochemical environments. The potential long‐term risk reduction associated with this approach requires a demonstration of causal relationships between sediment or contaminant stability on the one hand, and microbial reactivity on the other. The spatial analysis needed to fully understand and quantify these correlations requires sensitive probabilistic techniques. Geostatistics has been used for the characterization of multi‐scale spatial patterns for the last few decades, and the analysis of microbial attributes has shown significant spatial structures on microbial abundance and activity. However, there is a dearth of information on the applicability of geostatistics to quantitatively describe the interaction between the microorganisms and their environment. Using the Passaic River (NJ) dioxin data as a model dataset, multiple scaling models were applied to scale and interpolate sampled dioxin data and derive dechlorination signatures in sediments. Unlike conventional geostatistic tools that are based on the point‐to‐point spatial structures, the new multi‐scale model (M‐Scale) introduces a new framework for spatial analysis in which regional values at different scales are anchored by the correlations to each other. Spatial dioxin distributions and microbial dechlorination signatures were used as benchmarks for comparison of M‐Scale to ordinary kriging. The results from cross‐validation and jackknifing approaches applied to these datasets were analyzed and compared using Quantile‐Quantile (Q–Q) plots and reproduction coefficients. These plots indicated that the M‐Scale better preserves the local features of hotspots during data interpolation to a basin‐wide scale. Current efforts focus on mapping microbial abundance and respiratory competence in the Anacostia River, based on measurements at three different scales. The outcomes of this work will be used to develop an uncertainty‐based spatial decision tool for site remediation in this watershed using various capping strategies.

Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b.

The carboxyl‐terminal extension on fungal mitochondrial DNA polymerases: identification of a critical region of the enzyme from Saccharomyces cerevisiae

Fungal mitochondrial DNA (mtDNA) polymerases, in comparison to their metazoan counterparts, harbour unique carboxyl-terminal extensions (CTEs) of varying lengths and unknown function. To determine the essential regions of the 279 residue CTE of the yeast enzyme (Mip1p), several CTE-truncation variants were expressed in Saccharomyces cerevisiae. The respiratory competence of mip1delta175 cells, in which Mip1p lacks the C-terminal 175 residues, is indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351 and Mip1pdelta279 rapidly lose mtDNA. Approximately one in six mip1delta216 transformants grew on glycerol, albeit poorly. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and the rapid loss of mtDNA during fermentative, but not respiratory, growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. Comparison of this essential segment with the sequences of other fungal mtDNA polymerases revealed novel features shared among the mtDNA polymerases of the Saccharomycetales.

The Saccharomyces cerevisiae COQ10 Gene Encodes a START Domain Protein Required for Function of Coenzyme Q in Respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.

Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast

Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.

Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast

Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.

Training and Education Challenges for the Twenty-first Century: Respiratory Care Competency and Practice

In recent years, there has been a paradigm shift in the roles and responsibilities of respiratory therapists. Therapists increasingly are expected to design and implement respiratory care plans within the scope of protocols. Respiratory therapists also are expected to perform greater numbers of advanced procedures such as endotracheal intubation and arterial catheter insertion. The greatest impact on respiratory care practice is likely to be the increasing use of the principles of evidence-based medicine. Each of these factors will affect how respiratory therapists should be trained and educated in the twenty-first century.

Expression of mitochondrial release factor in relation to respiratory competence in yeast

Mitochondria have a single release factor that recognizes all stop codons in mRNAs. The yeast mitochondrial release factor, mRF1, is a protein of 43 kDa that emerges from its precursor by cleavage of a mitochondrial targeting sequence. mRF1 is localized exclusively in mitochondria, even when it is overproduced. A several-fold increase in mRF1 levels slightly inhibits the growth of wild-type cells on media containing a non-fermentable carbon source. A direct antisuppressor effect of overproduced mRF1 is observed, since the MRF1 gene on a multicopy plasmid causes Gly(-) phenotypes of the leaky mit(-) point mutations in mtDNA. We also examine steady-state mRF1 levels in a respiratory-deficient mrf1-780 mutant with inhibited mitochondrial translation. We show that both the mRF1 protein and the MRF1 transcript are elevated in mrf1-780 cells. A similar increase in mRF1 expression is observed in the rho(0) strain with no mitochondrial translation. This is indicative of retrograde signalling in the regulation of MRF1 expression. According to our hypothesis, inhibition of translation in the mrf1-780 strain is due to mitoribosome stalling at the stop codon and the observed elevated level of release factor is a secondary effect of respiratory deficiency.

Oxidative DNA Damage Causes Mitochondrial Genomic Instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Role of the Novel Metallopeptidase MoP112 and Saccharolysin for the Complete Degradation of Proteins Residing in Different Subcompartments of Mitochondria

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m- and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.

Aconitase Couples Metabolic Regulation to Mitochondrial DNA Maintenance

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.

Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining

Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA–protein complexes, termed nucleoids. The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht. We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen. Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells. We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2′,3′-dideoxycytidine. We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease. Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells. This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (ρ 0 cells) in culture even at very low numbers. We have also adapted the technique to efficiently sort ρ 0 cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence. This should be useful for the construction of new trans-mitochondrial ‘cybrid’ cell lines.

The Energetic Conversion Competence of Escherichia coli during Aerobic Respiration Studied by 31P NMR Using a Circulating Fermentation System

To determine the actual potential of the energetic conversion efficiency of Escherichia coli during aerobic respiration, apparent P/O ratios (P/O(app)) under either limited or standard glucose-feeding conditions were estimated. The previously reported circulating fermentation system (CFS) was used, and (31)P NMR saturation-transfer (ST) techniques were employed. By coupling with on-line NMR observations, CFS allowed us to evaluate cellular energetics directly, with both the dissolved oxygen tension and glucose feeding precisely controlled to prevent the effect of substrate-level phosphorylation based on aerobic or anaerobic acidogenesis in E. coli cells. Phosphate consumption rates under standard and limited glucose-conditions were estimated as 4.62 +/- 0.46 and 1.99 +/- 0.11 micromol/s g of dry cell weight (DCW), respectively. Using simultaneously assessed O(2) consumption rates, the P/O(app) values under these two conditions were estimated as 1.4 +/- 0.3 and 1.5 +/- 0.1, respectively. To correlate the obtained P/O(app) values with the potential efficiency of respiratory enzymes, we determined the activities of two NADH dehydrogenases (NDH 1 and 2) and two ubiquinol oxidases (bo- and bd-type) during the periods when ST was performed. NDH-1 activities in standard or limited glucose cultures were maintained at 57% or 58% of the total NADH oxidizing activity. The percentages of bo-type oxidase activity in relation to the total ubiqinol oxidizing activity under the standard and limited glucose conditions were 32% and 36%, respectively. These percentages of enzymatic activities represent the respiratory competence of E. coli cells, suggesting that, during the NMR observatory period, the enzymatic activity was not at a maximum, which could also explain the estimated P/O(app) values. If this is the case, enhancing the expression of the bo-type oxidase or disrupting of the bd-type oxidase gene could be effective approach to increasing both the P/O ratio and cellular yields.

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho 0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death.

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology.

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl-CoA dependent manner. We describe here the identification of the 3-hydroxyacyl-ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony-colour-sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non-fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology, were found to have a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase-isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3-hydroxyacyl-thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.

Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H+-translocating ATP Synthase of Saccharomyces cerevisiae

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

QO Site Deficiency Can Be Compensated by Extragenic Mutations in the Hinge Region of the Iron-Sulfur Protein in the bc1 Complex of Saccharomyces cerevisiae

The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(O)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(O) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 A from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 A from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(O) site and the ISP via the flexible hinge region and that fine-tuning of the Q(O) site catalysis can be achieved by subtle changes in the linker domain of the ISP.