Conformational coupling between the active site and residues within the KC-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K^C-channel is a conserved glutamate in subunit III. However, the majority of the K^C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K^C-channel, was found to depend on the conformation of Y241^Vc, located in subunit I at the interface with subunit III. Mutations of Y241^Vc (to A/F/H/S) in the Vibrio cholerae cbb₃ eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b₃. The data suggest a linkage between residues lining the K^C-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241^Vc and the active site cross-linked Y255^Vc, as well as two Cu_B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.Nearly all aerobic organisms rely on one or more heme-copper oxygen reductases (HCOs) as the terminal enzyme in their respiratory chains. These enzymes are virtually all proton pumps, coupling the generation of a proton motive force to the chemistry of reducing O₂ to water (1–3). Critical components of these enzymes include proton-conducting channels, which are conserved structures within each major family of HCOs (A, B, and C families) (4–6). These proton channels are defined by conserved polar residues, as well as internal water molecules that provide a hydrogen-bonded pathway for proton diffusion within the protein by a Grotthus-type mechanism (7–9). The channels are required to provide pathways both for chemical protons, which are consumed at the enzyme active site to form water, and for pumped protons, which are transported across the membrane. For prokaryotic A-family HCOs, there are two well-defined proton-input channels leading from the bacterial cytoplasm to the vicinity of the active site, designated the D-channel and the K-channel, respectively (10, 11).For each O₂ consumed, four chemical protons are taken from the bacterial cytoplasm and an additional four protons are taken from the cytoplasm and delivered to the periplasm, for Gram-negative organisms. Previous studies have demonstrated that all four of the pumped protons along with two chemical protons are taken up by the D-channel, whereas the K-channel is required for delivering two chemical protons to the active site during the portion of the catalytic cycle before the binding of O₂ (11). An H-channel has also been proposed for the mammalian cytochrome c oxidase (3), but with no equivalent in the prokaryotic or fungal HCOs.Surprisingly, the B- and C-families of oxygen reductases, which are found only in prokaryotes, each uses only one proton-input channel, located in the homologous part of the protein as the K-channel of the A-family oxygen reductases (6, 12, 13). We refer to these proton channels as the K^B- and K^C-channels for the B- and C-family enzymes, respectively. The patterns of conserved polar residues that define the K^B- and K^C-channels are unique to each family of enzymes, and both differ from the polar residues conserved within the K-channel of the A-family oxygen reductases. Because there is no equivalent of the D-channel in the B- and C-family oxygen reductases, all four of the chemical protons, as well as all pumped protons, must use the K^B- and K^C-channels. The present study explored the properties of mutations within the K^C-channel of cytochrome cbb₃ from Vibrio cholerae.The X-ray structure of the cytochrome cbb₃ (C-family oxygen reductase) from Pseudomonas stutzeri has confirmed the presence of only a single proton-conducting channel, located mostly within subunit I (CcoN) (13). Although the resolution is insufficient to define internal water molecules within the K^C-channel, molecular dynamics (MD) simulations have demonstrated that highly dynamic water wires can form within the K^C-channel and provide a pathway for proton translocation (14). Furthermore, these simulations suggest that the chemical and pumped protons share the same track but diverge after Y317^Ps (P. stutzeri)^*, at which point two pathways are possible, one for chemical protons leading to the active site and another for pumped protons leading to the propionates of heme b₃, which is near the periplasmic bulk aqueous phase.Although the bulk of the K^C-channel is located in subunit I (CcoN), mutagenesis studies of the cytochrome cbb₃ from Rhodobacter sphaeroides have demonstrated that the channel entrance is a highly conserved glutamate located on the cytoplasmic surface of subunit III (CcoP; E49^IIIPs) (15) (Fig. 1). The proton pathway from E49^IIIPs to the portion of the channel within subunit I (CcoN) is not evident. Here we used MD simulations to identify a hydrated pathway from the cytoplasmic solution and mediating direct contacts between E49^III and CcoN portion of the K^C-channel, supporting the role of E49^IIIPs in proton transfer. Site-directed mutagenesis studies of cytochrome cbb₃ from V. cholerae confirm the importance of the residues implicated as being part of the K^C-channel. An important finding is that a number of mutations within the K^C-channel also perturb the active site of the enzyme, suggesting a conformational coupling between the channel and the active site of the enzyme.Open in a separate windowFig. 1.Structure of cytochrome cbb₃ from P. stutzeri. (A) Ribbon structure with the CcoN in pink, CcoO in green, and CcoP in blue. Heme b and heme b₃ are highlighted along with the residues in the K^C-channel. (B) Enlargement of the region showing residues in the K^C-channel examined in the present study. The structure is that of PDB ID code 3MK7 from P. stutzeri (13), but the residue numbering is that from V. cholerae. Displayed water molecules were modeled using the DOWSER program (22).     

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb₃ cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb₃ cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.The known terminal oxidases according to the structure of their active centers and their phylogenetic relations are subdivided into two superfamilies (1). One is composed of numerous representatives containing a heme-copper binuclear active center (BNC). Oxidases belonging to the other superfamily have no copper. This superfamily includes bacterial oxidases of the bd type. The superfamily of representatives with heme-copper BNC is subdivided in turn into two groups, cytochrome c oxidases (Coxs) and quinol oxidases, depending upon the electron donor, which can be either cytochrome c or quinol. Quinol oxidases with a heme-copper BNC are found only in prokaryotes, whereas Coxs are widespread among living organisms of all domains: Eukarya (where they are found in mitochondria and chloroplasts), Bacteria, and Archaea. Although terminal oxidases with heme-copper BNC constitute a diverse group of multisubunit enzymes having from 2 to 13 subunits, conservatism and similar architecture are obviously inherent in their main (catalytic) subunit. The catalytic center of the main subunit always contains two hemes and copper as redox active prosthetic groups and a redox active tyrosine covalently bound to histidine in the polypeptide chain (2–5). Iron of one of the hemes and copper constitute the BNC. Coxs are the best-studied group of terminal oxidases. The basic mechanism of energy transduction by Coxs during respiration consists of the oxidation of cytochrome c by molecular oxygen (O₂) coupled to transmembrane pumping of protons (H⁺). This process results in reduction of O₂ to water by the BNC, where O₂ is bound. In Coxs, it requires four protons (“chemical” H⁺ for water production) taken from the inner side of the membrane and can be coupled to the translocation of another four protons (“pumped” H⁺) from the inner to the outer side of the membrane into the intermembrane or the periplasmic space of mitochondria or prokaryotic cells, respectively, according to the following equation (6–8):4cytc2++4Hin,chem++4Hin,chem++4Hin,pump++O2→4cytc3++4Hout,pump++2H2O.In A-type Coxs, two H⁺ pathways in the main subunit were identified, the so-called D channel, conducting all pumped and part of chemical H⁺, and the K channel, conducting most of chemical H⁺ (9). In C-type Coxs, only a K-channel analog was found (10). The described catalytic events are accomplished through generation of a transmembrane difference in H⁺ potentials (Δμ¯H+), which is used as a convertible membrane-linked biological currency. Microorganisms living in an alkaline environment maintain a nearly neutral cytoplasmic pH (11). This presents a problem for alkaliphiles because it gives rise to an inverted pH gradient that decreases the Δμ¯H+ (12, 13). Some alkaliphilic microorganisms solve this problem by using an Na⁺-pumping NADH-CoQ reductase (NQR) (14), and perhaps a Na⁺-pumping terminal oxidase, as was assumed (15). At present, NQR is the only respiratory chain enzyme for which Na⁺ pumping has been directly and undoubtedly established (16). However, NQR is absent in the extremely alkaliphilic bacterium Thioalkalivibrio versutus AL2, which inhabits an alkaline (∼pH 10) Siberian soda lake at saturating salt concentrations (17). T. versutus is a chemolithotroph that oxidizes sulfur compounds and employs Cox as a terminal component of its aerobic electron transport chain. Here we report that T. versutus uses a novel C-type Cox that (i) specifically requires Na⁺ for its activity and (ii) electrogenically exports Na⁺ from cells or right-side-out subcellular membrane vesicles, the process being coupled to oxidation of ascorbate by O₂.     

The mechanism of proton pumping by cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the “peroxy” forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a₃ propionates and are driven to the outside (“pumped”) by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P → F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F′ with an alternative route to O.     

A functional nitric oxide reductase model

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N₂O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme Fe_B does not show NO reduction activity. These results are consistent with a so-called “trans” mechanism for the reduction of NO by bacterial NOR.     

Coordination of {Mo142} Ring to La3+ Provides Elliptical {Mo134La10} Ring with a Variety of Coordination Modes

A 28-electron reduced C_2h-Mo-blue 34Ǻ outer ring diameter circular ring, [Mo₁₄₂O₄₂₉H₁₀(H₂O)₄₉(CH₃CO₂)₅(C₂H₅CO₂)]^30- (≡{Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}) comprising eight carboxylate-coordinated (with disorder) {Mo₂} linkers and six defect pockets in two inner rings (four and three for each, respectively), reacts with La³⁺ in aqueous solutions at pH 3.5 to yield a 28-electron reduced elliptical C_i-Mo-blue ring of formula [Mo₁₃₄O₄₁₆H₂₀(H₂O)₄₆{La(H₂O)₅}₄{La(H₂O)₇}₄{LaCl₂(H₂O)₅}₂]^10- (≡{Mo₁₃₄La₁₀}), isolated as the Na₁₀[Mo₁₃₄O₄₁₆H₂₀(H₂O)₄₆{La(H₂O)₅}₄{La(H₂O)₇}₄{LaCl₂(H₂O)₅}₂]·144 H₂O Na⁺ salt. The elliptical structure of {Mo₁₃₄La₁₀} showing 36 and 31 Å long and short axes for the outer ring diameters is attributed to four (A-D) modes of LaO₉/LaO₇Cl₂ tricapped-trigonal-prismatic coordination (TTP) geometries. Two different LaO₂(H₂O)₇ and one LaO₂(H₂O)₂Cl₂ TTP geometries (as A-C modes) for each of two inner rings result from the coordination of all three defect pockets of the inner ring for {Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}, and two LaO₄(H₂O)₅ TTP geometries (as D mode) result from the displacement of two (acetate/propionate-coordinated) binuclear {Mo₂} linkers with La³⁺ in each inner ring. The isothermal titration calorimetry (ITC) of the ring modification from circle to ellipsoid, showing the endothermic reaction of [La³⁺]/[{Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)}] = 6/1 with ΔH = 22 kJ⋅mol^-1, ΔS = 172 J⋅K^-1⋅mol^-1, ΔG = −28 kJ⋅mol^-1, and K = 9.9 × 10⁴ M^-1 at 293 K, leads to the conclusion that the coordination of the defect pockets to La³⁺ precedes the replacement of the {Mo₂} linkers with La³⁺. ¹³⁹La- NMR spectrometry of the coordination of {Mo₁₄₂(CH₃CO₂)₅(C₂H₅CO₂)} ring to La³⁺ is also discussed.     

Entrance of the proton pathway in cbb3-type heme-copper oxidases

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O(2) reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb(3)) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb(3) numbering) in the accessory subunit CcoP (E25(P)). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb(3) by O(2) is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25(P) as the entry point to the proton pathway in cbb(3) and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O(2) reduction. The cbb(3) is in addition to O(2) reduction capable of NO reduction, an activity that was largely retained in the E25(P) variants, consistent with a scenario where NO reduction in cbb(3) uses protons from the periplasmic side of the membrane.     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the "peroxy" state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH approximately 7.5. The movement of the Lys is proposed to regulate proton transfer by "shutting off" the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This "shutoff" prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.     

Inspecting Insulin Products Using Water Proton NMR. I. Noninvasive vs Invasive Inspection

Background:There is a clear need to transition from batch-level to vial/syringe/pen-level quality control of biologic drugs, such as insulin. This could be achieved only by noninvasive and quantitative inspection technologies that maintain the integrity of the drug product.Methods:Four insulin products for patient self-injection presented as prefilled pens have been noninvasively and quantitatively inspected using the water proton NMR technology. The inspection output is the water proton relaxation rate R₂(¹H₂O), a continuous numerical variable rather than binary pass/fail.Results:Ten pens of each product were inspected. R₂(¹H₂O) displays insignificant variation among the 10 pens of each product, suggesting good insulin content uniformity in the inspected pens. It is also shown that transferring the insulin solution out of and then back into the insulin pen caused significant change in R₂(¹H₂O), presumably due to exposure to O₂ in air.Conclusions:Water proton NMR can noninvasively and quantitatively inspect insulin pens. wNMR can confirm product content uniformity, but not absolute content. Its sensitivity to sample transferring provides a way to detect drug product tampering. This opens the possibility of inspecting every pen/vial/syringe by manufacturers and end-users.     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O₂ to H₂O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled (“gating” region) overlaps with a channel through which O₂ is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O₂ access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O₂ delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.     

Complete tracking of transient proton flow through active chloroplast ATP synthase

Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of P_i and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typically 1.3 H⁺ per CF1 and flash group. Proton flow was blocked by CF0-CF1 inhibitors: N,N′-dicyclohexylcarbodiimide, acting on the channel component CF0, and tentoxin, acting on the catalytic component CF1. The half-rise time was 40 ms in ¹H₂O and 78 ms in ²H₂O. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a K_m(P_i) of only 14 μM, contrasting with a K_m of several hundred micromolar for continuous ATP synthesis at high rate. This might reflect a resistance to P_i diffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CF0-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CF0-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.     

Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe3O4 Magnetic Nanoparticle Bio-Complex Film

A DNA/chitosan-Fe₃O₄ magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe₃O₄ nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe₃O₄/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe₃O₄ and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe₃O₄ film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s⁻¹. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe^(III)) and HRP(Fe^(II)). The obtained electrode also displayed an electrocatalytic reduction behavior towards H₂O₂. The resulting DNA/chitosan/Fe₃O₄/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm⁻²·M⁻¹) toward H₂O₂. A linear response to H₂O₂ measurement was obtained over the range from 2 μM to 100 μM (R² = 0.99) and an amperometric detection limit of 1 μM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.     

The Corrosion Behavior of Pure Iron under Solid Na2SO4 Deposit in Wet Oxygen Flow at 500 °C

The corrosion behavior of pure Fe under a Na₂SO₄ deposit in an atmosphere of O₂ + H₂O was investigated at 500 °C by thermo gravimetric, and electrochemical measurements, viz. potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and surface characterization methods viz. X-ray diffraction (XRD), and scanning electron microscope (SEM)/energy dispersive spectroscopy(EDS). The results showed that a synergistic effect occurred between Na₂SO₄ and O₂ + H₂O, which significantly accelerated the corrosion rate of the pure Fe. Briefly, NaFeO₂ was formed in addition to the customary Fe oxides; at the same time, H₂SO₄ gas was produced by introduction of water vapor. Subsequently, an electrochemical corrosion reaction occurred due to the existence of Na₂SO₄, NaFeO₂, and H₂O. When this coupled to the chemical corrosion reaction, the progress of the chemical corrosion reaction was promoted and eventually resulted in the acceleration of the corrosion of the pure Fe.     

HIV protease inhibitors elicit volume-sensitive Cl current in cardiac myocytes via mitochondrial ROS

HIV protease inhibitors (HIV PI) reduce morbidity and mortality of HIV infection but cause multiple untoward effects. Because certain HIV PI evoke production of reactive oxygen species (ROS) and volume-sensitive Cl⁻ current (I_Cl,swell) is activated by ROS, we tested whether HIV PI stimulate I_Cl,swell in ventricular myocytes. Ritonavir and lopinavir elicited outwardly rectifying Cl⁻ currents under isosmotic conditions that were abolished by the selective I_Cl,swell-blocker DCPIB. In contrast, amprenavir, nelfinavir, and raltegravir, an integrase inhibitor, did not modulate I_Cl,swell acutely. Ritonavir also reduced action potential duration, but amprenavir did not. I_Cl,swell activation was attributed to ROS because ebselen, an H₂O₂ scavenger, suppressed ritonavir- and lopinavir-induced I_Cl,swell. Major ROS sources in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin failed to block ritonavir- or lopinavir-induced currents, although it blocks I_Cl,swell elicited by osmotic swelling or stretch. In contrast, rotenone, a mitochondrial e⁻ transport inhibitor, suppressed both ritonavir- and lopinavir-induced I_Cl,swell. ROS production was measured in HL-1 cardiomyocytes with C-H₂DCFDA-AM and mitochondrial membrane potential (ΔΨ_m) with JC-1. Flow cytometry confirmed that ritonavir and lopinavir but not amprenavir, nelfinavir, or raltegravir augmented ROS production, and HIV PI-induced ROS production was suppressed by rotenone but not NADPH oxidase blockade. Moreover, ritonavir, but not amprenavir, depolarized ΔΨ_m. These data suggest ritonavir and lopinavir activated I_Cl,swell via mitochondrial ROS production that was independent of NADPH oxidase. ROS-dependent modulation of I_Cl,swell and other ion channels by HIV PI may contribute to some of their actions in heart and perhaps other tissues.     

O2 reduction by a functional heme/nonheme bis-iron NOR model complex

O₂ reactivity of a functional NOR model is investigated by using electrochemistry and spectroscopy. The electrochemical measurements using interdigitated electrodes show very high selectivity for 4e O₂ reduction with minimal production of partially reduced oxygen species (PROS) under both fast and slow electron flux. Intermediates trapped at cryogenic temperatures and characterized by using resonance Raman spectroscopy under single-turnover conditions indicate that an initial bridging peroxide intermediate undergoes homolytic OO bond cleavage generating a trans heme/nonheme bis-ferryl intermediate. This bis ferryl species can oxygenate 2 equivalents of a reactive substrate.     

A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized “as-isolated” form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529–533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-Å resolution by limiting the X-ray dose for each shot and by using many (≈400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O₂ reduction site (Fe³⁺-O⁻-O⁻-Cu²⁺), in contrast to a ferric hydroxide (Fe³⁺-OH⁻) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O₂ reduction site suppresses the proton pumping function.     

Initial Processes of Atomic Layer Deposition of Al2O3 on InGaAs: Interface Formation Mechanisms and Impact on Metal-Insulator-Semiconductor Device Performance

Interface-formation processes in atomic layer deposition (ALD) of Al₂O₃ on InGaAs surfaces were investigated using on-line Auger electron spectroscopy. Al₂O₃ ALD was carried out by repeating a cycle of Al(CH₃)₃ (trimethylaluminum, TMA) adsorption and oxidation by H₂O. The first two ALD cycles increased the Al KLL signal, whereas they did not increase the O KLL signal. Al₂O₃ bulk-film growth started from the third cycle. These observations indicated that the Al₂O₃/InGaAs interface was formed by reduction of the surface oxides with TMA. In order to investigate the effect of surface-oxide reduction on metal-insulator-semiconductor (MIS) properties, capacitors and field-effect transistors (FETs) were fabricated by changing the TMA dosage during the interface formation stage. The frequency dispersion of the capacitance-voltage characteristics was reduced by employing a high TMA dosage. The high TMA dosage, however, induced fixed negative charges at the MIS interface and degraded channel mobility.     

Synthesis,Crystal Structure and Luminescent Property of Mg(II) Complex with N-Benzenesulphonyl-L-Leucine and 1,10-Phenanthroline

A new complex [Mg(L)₂(phen)(H₂O)₂](phen)(H₂O)₂ [L= N-benzenesulphonyl-L-leucine] was synthesized by the reaction of magnesium chloride hexahydrate with N-benzenesulphonyl-L-leucine and 1,10-phenanthroline in the CH₃CH₂OH/H₂O (v:v = 5:1). It was characterized by elemental analysis, IR and X-ray single crystal diffraction analysis. The crystal of the title complex [Mg(L)₂(phen)(H₂O)₂](phen)(H₂O)₂ belongs to triclinic, space group P-1 with a = 0.72772(15) nm, b = 1.4279(3) nm, c = 1.4418(3) nm, α = 63.53(3)°, β = 79.75(3)°, γ = 81.83(3)°, V = 1.3163(5) nm³, Z =1, D_c= 1.258 μg·m⁻³, μ = 0.177 mm⁻¹, F(000) = 526, and final R₁ = 0.0506, ωR₂ = 0.1328. The complex comprises a six-coordinated magnesium(II) center, with a N₂O₄ distorted octahedron coordination environment. The molecules are connected by hydrogen bonds and π-π stacking to form one dimensional chain structure. The luminescent property of the Mg(II) complex has been investigated in solid.