The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245

Chronic granulomatous disease (CGD) is a rare inherited disorder associated with a profound predisposition to infection due to the lack of a microbicidal oxidase system in the phagocytes of these patients. This syndrome is most commonly inherited through a defect on the X chromosome and the only clearly defined component of the oxidase system, the very unusual cytochrome b (b-245), has been shown to be missing from the cells of these patients. This cytochrome is a heterodimer composed of an alpha-chain of relative molecular mass (Mr) 23,000 (23K) and a 76-92K beta-chain; neither are detectable in neutrophils from X-linked CGD subjects. The defective X-CGD gene has recently been cloned by 'reverse genetics' but the protein predicted from the proposed complementary DNA sequence was not identified. We have purified the beta-chain of the cytochrome and sequenced 43 amino acids from the N terminus. Almost complete homology was obtained between this sequence and that of the complementary nucleotides 19-147 of the sequence of the X-CGD gene, originally designated as a non-coding region.     

Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1

Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.     

Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare syndrome, found predominantly in male children and characterized by life-threatening, recurrent infections. The superoxide (O2-)/hydrogen peroxide (H2O2) generating system in the granulocytes and monocytes of CGD patients is completely defective. Furthermore, a novel type of cytochrome b, detected by the optical spectrum of phagocytes from healthy subjects, is lacking in those of most male CGD patients. In female CGD patients, the cytochrome b is present, but cannot, as in normal cells, be reduced on metabolic stimulation of the phagocytes in anaerobic conditions. Here, to demonstrate the importance of cytochrome b in this system and to investigate the genetic background of the various forms of CGD, we have hybridized monocytes from a cytochrome b negative, X-linked male CGD patient with monocytes from a cytochrome b positive, male CGD patient with unknown genetic background. Monocytes were used because they are the only blood phagocytes that show an active protein synthesis, whereas fibroblasts or lymphocytes do not express the O2-/H2O2 generating system. The heterologous hybrids were positive in the nitroblue tetrazolium (NBT) slide test, indicating the complementation of the O2-/H2O2 generating system, whereas the homologous hybrids remained negative, as did the non-fused cells of these patients. We thus conclude that cytochrome b is part of the O2-/H2O2 generating system and that somatic cell hybridization experiments with monocytes provide a means of studying the genetic background of CGD patients. We believe this to be the first report of genetic complementation by somatic cell hybridization experiments using monocytes instead of fibroblasts.     

The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex

The bacteriocidal capacity of phagocytic cells is impaired in X-linked chronic granulomatous disease (X-CGD), a disorder characterized by the absence of functional plasma-membrane-associated NADPH oxidase. The components of this oxidase system, their correspondence with specific genetic loci, and the primary protein defect in X-CGD remain incompletely defined. We recently reported cloning of the putative X-CGD gene on the basis of DNA linkage. To identify the predicted protein in vivo, antibodies were raised to a synthetic peptide derived from the complementary DNA sequence and to a fusion protein produced in Escherichia coli. In Western blots antisera detect a neutrophil protein of relative molecular mass in 90,000 (90K) that is absent in X-CGD patients. Antisera also react with the larger component of cytochrome b recently purified from neutrophil plasma membranes as a complex of glycosylated 90K and non-glycosylated 22K polypeptides. Based on our identification of the X-CGD protein in vivo, we propose that one of its critical roles is to interact with the 22K species to form a functional cytochrome b complex.     

Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

Pumping of protons from the mitochondrial matrix by cytochrome oxidase

The stoichiometry and mechanism of redox-linked proton translocation by the mitochondrial respiratory chain is a major issue of debate in membrane bioenergetics. The function of cytochrome oxidase is a focal point of disagreement. In 1977 it was suggested that the terminal component of the respiratory chain, cytochrome oxidase, functions as a redox-linked proton pump. That and subsequent studies were based mainly on measurements of proton ejection from mitochondria or from vesicles reconstituted with isolated cytochrome oxidase, or on measurements of translocation of electrical charge equivalents across mitochondrial and vesicle membranes. This proton-translocating function of cytochrome oxidase is confirmed here by a quantitative determination of proton uptake from the inside (matrix) of intact mitochondria.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

Functional relationship of cytochrome c(6) and plastocyanin in Arabidopsis

Photosynthetic electron carriers are important in converting light energy into chemical energy in green plants. Although protein components in the electron transport chain are largely conserved among plants, algae and prokaryotes, there is thought to be a major difference concerning a soluble protein in the thylakoid lumen. In cyanobacteria and eukaryotic algae, both plastocyanin and cytochrome c(6) mediate electron transfer from cytochrome b(6)f complex to photosystem I. In contrast, only plastocyanin has been found to play the same role in higher plants. It is widely accepted that cytochrome c(6) has been evolutionarily eliminated from higher-plant chloroplasts. Here we report characterization of a cytochrome c(6)-like protein from Arabidopsis (referred to as Atc6). Atc6 is a functional cytochrome c localized in the thylakoid lumen. Electron transport reconstruction assay showed that Atc6 replaced plastocyanin in the photosynthetic electron transport process. Genetic analysis demonstrated that neither plastocyanin nor Atc6 was absolutely essential for Arabidopsis growth and development. However, plants lacking both plastocyanin and Atc6 did not survive.     

A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.     

Identification of the electron transfers in cytochrome oxidase that are coupled to proton-pumping

Mitochondrial cytochrome oxidase is a functionally complex, membrane-bound respiratory enzyme which catalyses both the reduction of O2 to water and proton-pumping. During respiration, an exogenous donor, cytochrome c, donates four electrons to O2 bound at the bimetallic haem alpha 3 Fe-Cu centre within the enzyme. These four electron transfers are mediated by the enzyme's haem alpha and CuA redox centres and result in the translocation of four protons across the inner mitochondrial membrane. The molecular mechanism of proton translocation has not yet been delineated, however, and in the absence of direct experimental evidence all four electron transfers have been assumed to couple equally to proton-pumping. Here, I report the effects of proton-motive force and membrane potential on two equilibria involving intermediates of the bimetallic centre at different levels of O2 reduction. The results show that only two of the electron transfers, to the 'peroxy' and 'oxyferryl' intermediates of the bimetallic centre, are linked to proton translocation, a finding which strongly constrains candidate mechanisms for proton-pumping.     

Regulation of the arachidonic acid-stimulated respiratory burst in neutrophils by intracellular and extracellular calcium

The respiratory burst is an important physiological function of the neutrophils in killing the bacteria invading in human body. We used chemiluminescence method to measure the exogenous arachidonic acid-stimulated respiratory burst, and measured the cytosolic free calcium concentration in neutrophils by the fluorescence method. It was found that, on one hand, the arachidonic acid-stimulated respiratory burst was enhanced by elevating the cytosolic free calcium concentration in neutrophils with a potent endomembrane Ca2+-ATPase inhibitor, Thapsgargin; on the other hand, chelating the intracellular or extracellular calcium by EGTA or BAPTA inhibited the respiratory burst. Results showed that calcium plays an important regulatory role in the signaling pathway involved in the exogenous arachidonic acid-stimulated respiratory burst of neutrophils.     

The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen

Strict anaerobes cannot grow in the presence of greater than 5 micro M dissolved oxygen. Despite this growth inhibition, many strict anaerobes of the Bacteroides class of eubacteria can survive in oxygenated environments until the partial pressure of O2 (PO2) is sufficiently reduced. For example, the periodontal pathogens Porphyromonas gingivalis and Tannerella forsythensis colonize subgingival plaques of mammals, whereas several other Bacteroides species colonize the gastrointestinal tract of animals. It has been suggested that pre-colonization of these sites by facultative anaerobes is essential for reduction of the PO2 and subsequent colonization by strict anaerobes. However, this model is inconsistent with the observation that Bacteroides fragilis can colonize the colon in the absence of facultative anaerobes. Thus, this strict anaerobe may have a role in reduction of the environmental PO2. Although some strictly anaerobic bacteria can consume oxygen through an integral membrane electron transport system, the physiological role of this system has not been established in these organisms. Here we demonstrate that B. fragilis encodes a cytochrome bd oxidase that is essential for O2 consumption and is required, under some conditions, for the stimulation of growth in the presence of nanomolar concentrations of O2. Furthermore, our data suggest that this property is conserved in many other organisms that have been described as strict anaerobes.     

Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease

Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections. Various milestone discoveries have since been made, from an insufficient ability of patients' leucocytes to kill microbes to the underlying genetic abnormalities. In this inherited disorder, phagocytes lack NADPH oxidase activity and do not generate reactive oxygen species, most notably superoxide anion, causing recurrent bacterial and fungal infections. Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera. The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response. Here we show that a superoxide-dependent step in tryptophan metabolism along the kynurenine pathway is blocked in CGD mice with lethal pulmonary aspergillosis, leading to unrestrained Vgamma1(+) gammadelta T-cell reactivity, dominant production of interleukin (IL)-17, defective regulatory T-cell activity and acute inflammatory lung injury. Although beneficial effects are induced by IL-17 neutralization or gammadelta T-cell contraction, complete cure and reversal of the hyperinflammatory phenotype are achieved by replacement therapy with a natural kynurenine distal to the blockade in the pathway. Effective therapy, which includes co-administration of recombinant interferon-gamma (IFN-gamma), restores production of downstream immunoactive metabolites and enables the emergence of regulatory Vgamma4(+) gammadelta and Foxp3(+) alphabeta T cells. Therefore, paradoxically, the lack of reactive oxygen species contributes to the hyperinflammatory phenotype associated with NADPH oxidase deficiencies, through a dysfunctional kynurenine pathway of tryptophan catabolism. Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.     

Identification of the BAL-labile factor

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.     

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.     

A family of mitochondrial proteins involved in bioenergetICS and biogenesis

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.     

Association of a Ras-related protein with cytochrome b of human neutrophils

Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.     

Deficiency of the 50K dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy.

X-linked recessive Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoprotein. The dystrophin-glycoprotein complex has been proposed to span the sarcolemma to provide a link between the subsarcolemmal cytoskeleton and the extracellular matrix component, laminin. In DMD, the absence of dystrophin leads to a large reduction in all of the dystrophin-associated protein. We have investigated the possibility that a deficiency of a dystrophin-associated protein could be the cause of severe childhood autosomal recessive muscular dystrophy (SCARMD) with a DMD-like phenotype. Here we report the specific deficiency of the 50K dystrophin-associated glycoprotein (M(r) 50,000) in sarcolemma of SCARMD patients. Therefore, the loss of this glycoprotein is a common denominator of the pathological process leading to muscle cell necrosis in two forms of muscular dystrophy, DMD and SCARMD.