Subunits Of Cytochrome Research Articles

Three nucleus-encoded subunits of mitochondrial cytochrome c oxidase of the whiteleg shrimp Litopenaeus vannamei: cDNA characterization, phylogeny and mRNA expression during hypoxia and reoxygenation

The mitochondrial cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water playing a key role in the respiratory chain and ATP synthesis. The nucleus-encoded COX subunits do not participate in catalysis, but some are known to play a role in the expression, assembly and activity of the enzyme. Since hypoxia continuously affects the shrimp environment, it is important to study COX to understand their ability to deal with low oxygen levels. The goal of this research was to characterize the complementary DNA (cDNA) sequences of three nucleus-encoded subunits -coxIV, coxVa, and coxVb- and to evaluate the shrimp COX response to hypoxia by measuring their gene expression. The cDNA sequence of coxIV consisted of 532bp, which encodes a 17.47kDa protein, while coxVa cDNA consisted of 460bp and coded a protein of 17.11kDa, and the coxVb coding sequence consisted of 364bp encoding a 13.74kDa protein. Shrimp subunits do not have isoforms, and they are not differentially expressed during hypoxia, as observed in mammals. Coordinated changes were detected in the mRNA amounts of nuclear and mitochondrial subnits; these changes, at the transcriptional level, are suggested to be controlled through transcriptional factors Sp1 and NRF2.

Behavioral, Ecological and Genetic Differentiation in an Open Environment—A Study of a Mysid Population in the Baltic Sea

Diel vertical migration (DVM) is often assumed to encompass an entire population. However, bimodal nighttime vertical distributions have been observed in various taxa. Mysid shrimp populations also display this pattern with one group concentrated in the pelagia and the other near the bottom. This may indicate alternative migratory strategies, resembling the seasonal partial migrations seen in birds, fishes and amphibians, where only a subset of the population migrates. To assess the persistence of these alternative strategies, we analyzed the nitrogen and carbon stable isotope signatures (as proxies for diet), biochemical indices (as proxies for growth condition), and genetic population divergence in the Baltic mysid Mysis salemaai collected at night in the pelagia and close to the bottom. Stable isotope signatures were significantly different between migrants (pelagic samples) and residents (benthic samples), indicating persistent diet differences, with pelagic mysids having a more uniform and carnivorous diet. Sequencing of the mitochondrial cytochrome subunit I (COI) gene showed genetic differentiation attributable to geographic location but not between benthic and pelagic groups. Divergent migration strategies were however supported by significantly lower gene flow between benthic populations indicating that these groups have a lower predisposition for horizontal migrations compared to pelagic ones. Different migration strategies did not convey measurable growth benefits as pelagic and benthic mysids had similar growth condition indices. Thus, the combination of ecological, biochemical and genetic markers indicate that this partial migration may be a plastic behavioral trait that yields equal growth benefits.

Essentiality of respiratory activity for pentose utilization in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042

By random integrative mutagenesis with a kanMX4 cassette in Kluyveromyces marxianus DMKU 3-1042, we obtained three mutants of COX15, ATP25 and CYC3 encoding a cytochrome oxidase assembly factor (singleton), a transcription factor required for assembly of the Atp9p subunit of mitochondrial ATP synthase and cytochrome c heme lyase, respectively, as mutants lacking growth capability on xylose and/or arabinose. They exhibited incapability of growth on non-fermentable carbon sources, such as acetate or glycerol, and thermosensitiveness. Their biomass formation in glucose medium was reduced, but ethanol yields were increased with a high ethanol level in the medium, compared to those of the parental strain. Experiments with respiratory inhibitors showed that cox15 and cyc3, but not atp25, were able to grow in glucose medium containing antimycin A and that the atp25 mutant was KCN-resistant. Activities of NADH and ubiquinol oxidases in membrane fractions of each mutant became a half of that of the parent and negligible, respectively, and their remaining NADH oxidase activities were found to be resistant to KCN. Absolute absorption spectral analysis revealed that the peak corresponding to a + a 3 was very small in atp25 and negligible in cox15 and cyc3. These findings suggest that the K. marxianus strain possesses an alternative KCN-resistant oxidase that is located between primary dehydrogenases and the ubiquinone pool and that the respiratory activity is essential for utilization of pentoses.

Computational Quantification of the Physicochemical Effects of Heme Distortion: Redox Control in the Reaction Center Cytochrome Subunit of Blastochloris viridis

A facile, experimentally calibrated computational procedure is described that affords the relative ordering of heme cofactor reduction potentials with respect to intrinsic shifts brought about by apoprotein induced heme-macrocycle distortion. The method utilizes heme-Fe partial atomic charges and is useful with the computationally inexpensive B3LYP/3-21g method calculated for simplified heme models extracted from the Protein Data Bank incorporating only the effects of varying macrocycle conformations and thereby delineating their physicochemical effects. The procedure was successfully calibrated using the atomic coordinates and published midpoint potentials from the heme cofactors in wild-type and a series of heme-NO and -O(2) binding domain mutants and thus confirmed the sole conformational modulation of the redox potentials in these complexes. This technique was also applied to the reaction center tetraheme cytochrome subunit of Blastochloris viridis to build upon previous work elucidating the role that conformational control plays in photosynthetic systems, and it was found that this effect may account for up to 70% (54mv) of the observed differences in the reduction potentials of the four hemes. We validate the approach using larger basis sets up to and including the triple-ζ, doubly polarized and augmented 6-311+g** basis and discuss the specific conformational origins of the effect.

Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism 937ACC>937GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.

Astaxanthin protects against MPP+-induced oxidative stress in PC12 cells via the HO-1/NOX2 axis

BackgroundAlthough the etiology of PD remains unclear, increasing evidence has shown that oxidative stress plays an important role in its pathogenesis and that of other neurodegenerative disorders. NOX2, a cytochrome subunit of NOX, transports electrons across the plasma membrane to generate ROS, leading to physiological and pathological processes. Heme oxygenase-1 (HO-1) can be rapidly induced by oxidative stress and other noxious stimuli in the brain or other tissues. Astaxanthin (ATX), a carotenoid with antioxidant properties, is 100–1000 times more effective than vitamin E. The present study investigated the neuroprotective effects of ATX on MPP+-induced oxidative stress in PC12 cells.ResultsMPP+ significantly decreased MTT levels in a concentration-dependent manner. Hemin, SnPPIX and ATX didn’t exhibit any cytotoxic effects on PC12 cells. Pretreatment with ATX (5, 10, 20 μM), caused intracellular ROS production in the MPP+ group to decrease by 13.06%, 22.13%, and 27.86%, respectively. MPP+ increased NOX2, NRF2 and HO-1 protein expression compared with control (p < 0.05). Co-treatment with hemin or ATX suppressed NOX2 expression (p < 0.01), and greatly increased NRF2 and HO-1 expression (p < 0.01). MPP+ treatment up-regulated both NOX2 (p < 0.01) and HO-1 (p < 0.01) mRNA levels. Co-treatment with hemin or ATX significantly increased HO-1 mRNA levels (p < 0.01), and decreased NOX2 mRNA levels (p < 0.01). MPP+ increased NOX2 and HO-1 expression with considerable fluorescence extending out from the perinuclear region toward the periphery; this was attenuated by DPI. Co-treatment with hemin or ATX significantly up-regulated HO-1 expression and decreased NOX2 expression with considerable fluorescence intensity (stronger than the control and MPP+ groups).ConclusionsATX suppresses MPP+-induced oxidative stress in PC12 cells via the HO-1/NOX2 axis. ATX should be strongly considered as a potential neuroprotectant and adjuvant therapy for patients with Parkinson’s disease.

Peroxiredoxin 6 Enhances Phagocyte NADPH Oxidase (phox) Activity in Response to Agonists Acting Via Cell Surface Receptors and Intracellular Signaling Pathways.

Abstract 2139 IntroductionThe phagocyte NADPH oxidase (phox) is a multiprotein, transmembrane-enzyme-complex found in diverse professional phagocytes including neutrophils. Phox in its inactive state consists of the membrane-associated subunit cytochrome b558 (a dimer of gp91phox/Nox2 and p22phox) and cytoplasmic subunits which include p47phox, p67phox, p40phox and Rac1 or Rac2. Phosphorylation of phox proteins promotes tight interaction of the cytoplasmic phox components with cytochrome b558 and the resulting active complex catalyzes transmembrane electron transfer from cytoplasmic NADPH to molecular oxygen forming superoxide anion (O2−). O2− and other reactive oxygen species formed from it are microbicibal and crucial to pathogen killing by neutrophils.Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with a peroxiredoxin activity that reduces oxidized lipids and H2O2 and a PLA2 activity that cleaves the sn2 position of phospholipids forming the corresponding free fatty acid and lysophospholipid.Previous work in our laboratory has identified Prdx6 as a binding partner of p67phox and as an enhancer of O2− production by active phox; we have shown that this enhancement is due to products of the PLA2 activity of the peroxiredoxin but the precise mechanism by which these molecules act is unclear. Here we present evidence that Prdx6 causes maximal activation of phox when the complex is activated by physiological agonists (the formylated peptide fMLP and the particulate agonist serum opsonized zymosan (SOZ)) but not when it is activated by the non-physiological agonist phorbol 12-myristate 13-acetate (PMA). Because fMLF and SOZ interact with cell surface receptors but PMA enters the cell and activates protein kinase C (PKC) enzymes that directly phosphorylate phox proteins our data provides a clue as to the mode of action of Prdx6 in phox activation. MethodsPLB985 cells were used to model neutrophils. This myeloid cell line can be terminally differentiated to a neutrophil like state with phox activity by exposure to DMSO. Prdx6 was suppressed in these cells by stable expression of an shRNA resulting in an approximately 70% reduction of the protein. Following maturation of the cells they were stimulated with fMLP, SOZ or PMA and O2− production was measured by superoxide dismutase (SOD) inhibitable luminescence of Diogenes fluorophore. For fMLP, due to the transient nature of the O2- production, the entire amount of O2− production was measured. SOZ and PMA induced prolonged (>1hr) O2− production and O2− production over 1hr was thus measured. ResultsIn cells with Prdx6 suppressed, SOD inhibitable O2− production in response to fMLP was reduced by 53% ± 3% (SEM), n=34, which was significant (p<0.001). O2− produced in response to SOZ was reduced by 37% ± 9% (SEM), n = 4, which was also significant (p<0.05).In contrast, the response to PMA was not altered (p>0.5, n of at least 4) at a range of PMA concentrations from one causing slight activation of phox (10 ng/ml) to one causing maximal O2- production (1 μg/ml). ConclusionsfMLP binds to receptors on neutrophils and triggers intracellular signaling cascades that ultimately lead to phosphorylation and activation of phox. Similarly, opsonins of particulate stimuli bind cell surface complement receptors that trigger intracellular signaling cascades that activate phox. PMA on the other hand enters cells and activates PKC enzymes that directly phosphorylate phox proteins; it thus bypasses the physiological signaling pathways that mediate the fMLP and SOZ response.Since Prdx6 enhances the SOZ and fMLF mediated phox response but not the PMA mediated response we propose that in myeloid cells, products of the PLA2 activity of Prdx6 enhance the SOZ and PMA mediated signaling cascades. This may be via activation of signaling molecules in these pathways which would be consistent with the known ability of the fatty acid arachidonate to activate PKC enzymes, MAP kinases, PLA2 enzymes and a class Ia phosphatidylinositol 3-kinase and to mobilize Ca2+. Disclosures:No relevant conflicts of interest to declare.

Mutations in COX7B Cause Microphthalmia with Linear Skin Lesions, an Unconventional Mitochondrial Disease

Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation ofthe COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.

The Cbp3–Cbp6 complex coordinates cytochrome b synthesis with bc1 complex assembly in yeast mitochondria

Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc(1) complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc(1) complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc(1) complex assembly.

TAp73 depletion accelerates aging through metabolic dysregulation

Aging is associated with impaired scavenging of reactive oxygen species (ROS). Here, we show that TAp73, a p53 family member, protects against aging by regulating mitochondrial activity and preventing ROS accumulation. TAp73-null mice show more pronounced aging with increased oxidative damage and senescence. TAp73 deletion reduces cellular ATP levels, oxygen consumption, and mitochondrial complex IV activity, with increased ROS production and oxidative stress sensitivity. We show that the mitochondrial complex IV subunit cytochrome C oxidase subunit 4 (Cox4i1) is a direct TAp73 target and that Cox4i1 knockdown phenocopies the cellular senescence of TAp73-null cells. Results indicate that TAp73 affects mitochondrial respiration and ROS homeostasis, thus regulating aging.

Using population genetic methods to identify the origin of an invasive population and to diagnose cryptic subspecies ofTelchin licus(Lepidoptera: Castniidae)

Telchin licus, the giant sugarcane borer, is an important pest species of sugarcane in northeast Brazil. Four subspecies of Telchin licus are recognized in Brazil based on their geographic distribution and subtle differences in wing colour pattern. Some taxa are morphologically indistinguishable, and their accurate identification is key to their efficient control. Mitochondrial genes sequences (cytochrome oxidase I and subunit 6 of the nicotinamide adenine dinucleotide dehydrogenase) were applied to delimit taxonomic entities of T. licus, and to infer the origin of a newly established population in the state of São Paulo. The molecular data indicated that specimens sampled at different regions in Brazil are morphologically cryptic but genetically isolated entities, and at least three subspecies were assigned to the sampled localities. These data also suggested that the population collected from the state of São Paulo must have a common origin with populations from northeast Brazil, which corroborate the hypothesis that ornamental plants infested with larvae of T. licus might have been transported from the northeast to the southeast regions.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

BackgroundConstitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX).ResultsWe found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice.ConclusionTaken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today.

Применение молекулярных методов в систематике молей-чехлоносок (LEPIDOPTERA, COLEOPHORIDAE)

Discussed suprageneric system casebearer moths from a study of the nucleotide sequence of the gene first subunit of cytochrome c oxidase (COI). Confirmed the monophyly of the family and the availability sister Coleophoridae groups – family Batracherdridae, which are discussed in the rank of individual families. The proposed scheme, in general, supports the families of moths casebearer with selected earlier suprageneric taxa domestic and foreign experts, lepidopterist [1–5] based on morphological characteristics and features of these trophic caterpillars of moths.

Genetic Studies of the Drosophila Eye Link Energy Metabolism with Cell-Cycle Progression

Genetic studies of Drosophila eye imaginal disc development have provided a classic approach to dissect the coordination of cell proliferation and differentiation. This paper describes the characterization of a mutation in the tenured gene, which was recovered from a large-scale genetic screen based on its glossy eye phenotype. The authors show that this phenotype arises from a specific block in the G1 phase of the cell cycle, with no apparent effects on cell viability or differentiation. The remarkable part of this paper lies in the discovery that tenured encodes a subunit of cytochrome c oxidase, a key component in the electron transport chain. The authors show that the reduced ATP levels in tenured mutant cells leads to activation of the energy sensor AMPK that, in turn, acts through a p53 signaling pathway to reduce levels of the cell-cycle regulator Cyclin E. This paper provides, for the first time, a link between cellular energy levels and cell-cycle progression, giving us a framework to understand how metabolism and proliferation are coordinated during development. This PaperPick refers to Mitochondrial Regulation of Cell Cycle Progression during Development as Revealed by the tenured Mutation in Drosophila , by S. Mandal, P. Guptan, E. Owusu-Ansah, and U. Banerjee, published in December 2005. Video Abstract Utpal Banerjee discusses the findings of his group's 2005 study and the progress that has been made since.

Schizophrenia: Maternal inheritance and heteroplasmy of mtDNA mutations

Role of mitochondrial pathology in schizophrenia has not been fully clarified. We searched for distinctive variants in mtDNA extracted from the gray matter of postmortem brains and from peripheral blood samples. We screened mtDNA region containing 5 genes encoding subunits of cytochrome c oxidase and ATPases. Polymorphisms not already reported in databases are recorded as unregistered rare variants. Four unregistered, non-synonymous rare variants were detected in 4 schizophrenic samples. Seven registered non-synonymous variants were not previously detected in non-psychotic Japanese samples registered in the mtSNP database. These variants may contribute to disease pathophysiology. In one family, compound mutations showed co-segregation with schizophrenia. MtDNA mutations could confer a risk for schizophrenia in the Japanese population, although further analyses are needed.

X-ray structure of the dimeric cytochrome bc1 complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

The respiratory cytochrome bc1 complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c1 changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-Å resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c1 shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c1 surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c1 and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c552, the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.

Spectroscopic Characterization of the Molybdenum Cofactor of the Sulfane Dehydrogenase SoxCD from Paracoccus pantotrophus

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

The Putative Assembly Factor CcoH Is Stably Associated with the cbb 3 -Type Cytochrome Oxidase

Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb(3)-Cox stability. Biogenesis of cbb(3)-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb(3)-Cox. Most bacteria expressing cbb(3)-Cox also contain the ccoGHIS genes, which encode putative cbb(3)-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb(3)-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (N(out)-C(in)) topology. In its absence, neither the fully assembled cbb(3)-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb(3)-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb(3)-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb(3)-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb(3)-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb(3)-Cox than an assembly factor per se.