A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Aqp5-/- mice exhibit reduced maximal body O2 consumption under cold exposure, normal pulmonary gas exchange, and impaired formation of brown adipose tissue.

The fundamental body functions that determine maximal O2 uptake (V̇o2max) have not been studied in Aqp5-/- mice (aquaporin 5, AQP5). We measured V̇o2max to globally assess these functions and then investigated why it was found altered in Aqp5-/- mice. V̇o2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o2max reduced in Aqp5-/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5-/- mice. Lung function parameters as well as arterial O2 saturation were normal and identical between Aqp5-/- and WT mice, indicating that AQP5 does not contribute to pulmonary O2 exchange. The cause for the decreased V̇o2max thus might be found in decreased O2 consumption of an intensely O2-consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5-/- animals. We conclude that lack of AQP5 does not affect pulmonary O2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o2max under this condition.

Visualizing the gas channel of a monofunctional carbon monoxide dehydrogenase.

Carbon monoxide dehydrogenase (CODH) plays an important role in the processing of the one­carbon gases carbon monoxide and carbon dioxide. In CODH enzymes, these gases are channeled to and from the Ni-Fe-S active sites using hydrophobic cavities. In this work, we investigate these gas channels in a monofunctional CODH from Desulfovibrio vulgaris, which is unusual among CODHs for its oxygen-tolerance. By pressurizing D. vulgaris CODH protein crystals with xenon and solving the structure to 2.10 Å resolution, we identify 12 xenon sites per CODH monomer, thereby elucidating hydrophobic gas channels. We find that D. vulgaris CODH has one gas channel that has not been experimentally validated previously in a CODH, and a second channel that is shared with Moorella thermoacetica carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). This experimental visualization of D. vulgaris CODH gas channels lays groundwork for further exploration of factors contributing to oxygen-tolerance in this CODH, as well as study of channels in other CODHs. We dedicate this publication to the memory of Dick Holm, whose early studies of the Ni-Fe-S clusters of CODH inspired us all.

Carbon dioxide transport across membranes.

Carbon dioxide (CO2) movement across cellular membranes is passive and governed by Fick's law of diffusion. Until recently, we believed that gases cross biological membranes exclusively by dissolving in and then diffusing through membrane lipid. However, the observation that some membranes are CO2 impermeable led to the discovery of a gas molecule moving through a channel; namely, CO2 diffusion through aquaporin-1 (AQP1). Later work demonstrated CO2 diffusion through rhesus (Rh) proteins and NH3 diffusion through both AQPs and Rh proteins. The tetrameric AQPs exhibit differential selectivity for CO2 versus NH3 versus H2O, reflecting physico-chemical differences among the small molecules as well as among the hydrophilic monomeric pores and hydrophobic central pores of various AQPs. Preliminary work suggests that NH3 moves through the monomeric pores of AQP1, whereas CO2 moves through both monomeric and central pores. Initial work on AQP5 indicates that it is possible to create a metal-binding site on the central pore's extracellular face, thereby blocking CO2 movement. The trimeric Rh proteins have monomers with hydrophilic pores surrounding a hydrophobic central pore. Preliminary work on the bacterial Rh homologue AmtB suggests that gas can diffuse through the central pore and three sets of interfacial clefts between monomers. Finally, initial work indicates that CO2 diffuses through the electrogenic Na/HCO3 cotransporter NBCe1. At least in some cells, CO2-permeable proteins could provide important pathways for transmembrane CO2 movements. Such pathways could be amenable to cellular regulation and could become valuable drug targets.

Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen.

Hydrogenases are metalloenzymes that catalyse both H2 evolution and uptake. They are gas-processing enzymes with deeply buried active sites, so the gases diffuse through channels that connect the active site to the protein surface. The [NiFeSe] hydrogenases are a special class of hydrogenases containing a selenocysteine as a nickel ligand; they are more catalytically active and less O2-sensitive than standard [NiFe] hydrogenases. Characterisation of the channel system of hydrogenases is important to understand how the inhibitor oxygen reaches the active site to cause oxidative damage. To this end, crystals of Desulfovibrio vulgaris Hildenborough [NiFeSe] hydrogenase were pressurized with krypton and oxygen, and a method for tracking labile O2 molecules was developed, for mapping a hydrophobic channel system similar to that of the [NiFe] enzymes as the major route for gas diffusion.

Single-cell O2 exchange imaging shows that cytoplasmic diffusion is a dominant barrier to efficient gas transport in red blood cells.

Disorders of oxygen transport are commonly attributed to inadequate carrying capacity (anemia) but may also relate to inefficient gas exchange by red blood cells (RBCs), a process that is poorly characterized yet assumed to be rapid. Without direct measurements of gas exchange at the single-cell level, the barriers to O2 transport and their relationship with hematological disorders remain ill defined. We developed a method to track the flow of O2 in individual RBCs by combining ultrarapid solution switching (to manipulate gas tension) with single-cell O2 saturation fluorescence microscopy. O2 unloading from RBCs was considerably slower than previously estimated in acellular hemoglobin solutions, indicating the presence of diffusional barriers in intact cells. Rate-limiting diffusion across cytoplasm was demonstrated by osmotically induced changes to hemoglobin concentration (i.e., diffusive tortuosity) and cell size (i.e., diffusion pathlength) and by comparing wild-type cells with hemoglobin H (HbH) thalassemia (shorter pathlength and reduced tortuosity) and hereditary spherocytosis (HS; expanded pathlength). Analysis of the distribution of O2 unloading rates in HS RBCs identified a subpopulation of spherocytes with greatly impaired gas exchange. Tortuosity imposed by hemoglobin was verified by demonstrating restricted diffusivity of CO2, an acidic gas, from the dissipative spread of photolytically uncaged H+ ions across cytoplasm. Our findings indicate that cytoplasmic diffusion, determined by pathlength and tortuosity, is a major barrier to efficient gas handling by RBCs. Consequently, changes in RBC shape and hemoglobin concentration, which are common manifestations of hematological disorders, can have hitherto unrecognized and clinically significant implications on gas exchange.

Computational model of electrode-induced microenvironmental effects on pH measurements near a cell membrane.

The mechanism of gas transport across cell membranes remains a topic of considerable interest, particularly regarding the extent to which lipids vs. specific membrane proteins provide conduction pathways. Studies of transmembrane (CO2) transport often rely on data collected under controlled conditions, using pH-sensitive microelectrodes at the extracellular surface to record changes due to extracellular CO2 diffusion and reactions. Although recent detailed computational models can predict a qualitatively correct behavior, a mismatch between the dynamical ranges of the predicted and observed pH curves raises the question whether the discrepancy may be due to a bias introduced by the pH electrode itself. More specifically, it is reasonable to ask whether bringing the electrode tip near or in contact with the membrane creates a local microenvironment between the electrode tip and the membrane, so that the measured data refer to the microenvironment rather than to the free surface. Here, we introduce a detailed computational model, designed to address this question. We find that, as long as a zone of free diffusion exists between the tip and the membrane, the microenvironment behaves effectively as the free membrane. However, according to our model, when the tip contacts the membrane, partial quenching of extracellular diffusion by the electrode rim leads to a significant increase in the pH dynamics under the electrode, matching values measured in physiological experiments. The computational schemes for the model predictions are based on semi-discretization by a finite-element method, and an implicit-explicit time integration scheme to capture the different time scales of the system.

Tailorable Metal-Ceramic (Cu-TiC0.5) Layered Electrode with High Mechanical Property and Conductivity.

Two-dimensional materials have been extensively investigated in the fields of electrochemical sensors, field-effect transistors, and other electronic devices due to their large surface areas, high compatibility with device integration, and so on. Conventional electrodes, such as precious metal layers that are deposited on polymer or silicon wafers, have gradually revealed increasing difficulties in adapting to various device structures, especially for two-dimensional materials, which prefer high exposure of surface atoms. Here, we demonstrate a tailorable metal-ceramic (Cu-TiC0.5) layered structure as novel electrodes with high mechanical property and conductivity and fabricate a highly sensitive gas sensor with graphene lying on this proposed electrodes. The Cu-TiC0.5 layered structure exhibits remarkably high tensile yield strength and compressive yield strength, which increase 7 and 8 times than those of the pure copper, respectively. Meanwhile, excellent flexibility and conductivity could also be obtained with the further thinning of the Cu-TiC0.5 layered composite, which shows its potential applications in flexible electronics. Finally, we demonstrated that a graphene-based gas sensor fabricated on tailored metal-ceramic electrodes was ultrasensitive and robust, which benefits from the good thermal conductivity and peculiar gas channels etched on the surface of copper alloy electrodes.

CO2 Permeability of Rat Hepatocytes and Relation of CO2 Permeability to CO2 Production.

BACKGROUND/AIMS: It has been described that cells in culture with very low oxidative metabolism possess a low CO2 membrane permeability, PCO2, of ∼ 0.01 cm/s. On the other hand, cardiomyocytes and mitochondria with extremely high rates of O2 consumption exhibit very high CO2 membrane permeabilities of 0.1 and 0.3 cm/s, repectively. To ascertain that this represents a systematic relationship, we determine here PCO2 of hepatocytes, which exhibit an intermediate rate of O2 consumption. METHODS: We isolated intact hepatocytes with vitalities of ∼ 70% from rat liver and measured their CO2 permeability by the previously published mass spectrometric 18O exchange technique. RESULTS: We find a PCO2 of hepatocytes of 0.03 cm/s in the presence of FC5-208A and verapamil. FC5-208A was necessary to inhibt extracellular carbonic anhydrase, and verapamil was necessary to inhibit intracellular uptake of FC5-208A by the organic cation transporter OCT1 of hepatocytes. CONCLUSION: Rat hepatocytes with their intermediate rate of oxygen consumption also possess an intermediate CO2 permeability. From pairs of data for five types of cells/organelles, we find an excellent positive linear correlation between PCO2 and metabolic rate, suggesting an adaptation of PCO2 to the rate of O2 consumption.

Membranes with fast and selective gas-transport channels of laminar graphene oxide for efficient CO2 capture.

Graphene oxide (GO) nanosheets were engineered to be assembled into laminar structures having fast and selective transport channels for gas separation. With molecular-sieving interlayer spaces and straight diffusion pathways, the GO laminates endowed as-prepared membranes with excellent preferential CO2 permeation performance (CO2 permeability: 100 Barrer, CO2/N2 selectivity: 91) and extraordinary operational stability (>6000 min), which are attractive for implementation of practical CO2 capture.

Rapid CO2 permeation across biological membranes: implications for CO2 venting from tissue.

The degree to which cell membranes are barriers to CO2 transport remains controversial. Proteins, such as aquaporins and Rh complex, have been proposed to facilitate CO2 transport, implying that the nonchannel component of membranes must have greatly reduced CO2 permeability. To determine whether membrane CO2 permeation is rate limiting for gas transport, the spread of CO2 across multicellular tissue growths (spheroids) was measured using intracellular pH as a spatial readout. Colorectal HCT116 cells have basal water and NH3 permeability, indicating the functional absence of aquaporins and gas channels. However, CO2 diffusivity in HCT116 spheroids was only 24 ± 4% lower than in pure water, which can be accounted for fully by volume exclusion due to proteins. Diffusivity was unaffected by blockers of aquaporins and Rh complex (Hg(2+), p-chloromercuribenzoic acid, and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid) but decreased under hypertonic conditions (by addition of 300 mOsm mannitol), which increases intracellular protein crowding. Similar CO2 diffusivity was measured in spheroids of T47D breast cells (basal water permeability) and NHDF-Ad fibroblasts (aquaporin-facilitated water permeability). In contrast, diffusivity of NH3, a smaller but less lipophilic gas, was considerably slower than in pure water, as expected from rate-limiting membrane permeation. In conclusion, membranes, even in the functional absence of proposed gas channels, do not restrict CO2 venting from tissue growths.-Hulikova, A., Swietach, P. Rapid CO2 permeation across biological membranes: implications for CO2 venting from tissue.

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods.

We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last 100 years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2-as well as other gases-permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of "CO2-impermeable membranes" can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favor of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the (18)O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method.

Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel.

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability (Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I(-) > NO3(-) ≅ NO2(-) > Br(-) > Cl(-) > HCO3(-) > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9.

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances.