Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals

To detect changes in the oxygen concentration during biochemical reactions, the exchange broadening in the ESR spectra of nitroxide radicals caused by the dissolved oxygen, has been used. The measurements have been carried out using changes in the width either of the proton hyperfine structure components or of the nitrogen hyperfine structure line with an unresolved proton structure. Detection of mitochondrial respiration in a volume of about 10^?3 cm³ and respiration for 100±5 liver cells in a volume of about 10^?4 cm³ has been carried out.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

Density functional calculations on model tyrosyl radicals.

A gradient-corrected density functional theory approach (PWP86) has been applied, together with large basis sets (IGLO-III), to investigate the structure and hyperfine properties of model tyrosyl free radicals. In nature, these radicals are observed in, e.g., the charge transfer pathways in photosystem II (PSII) and in ribonucleotide reductases (RNRs). By comparing spin density distributions and proton hyperfine couplings with experimental data, it is confirmed that the tyrosyl radicals present in the proteins are neutral. It is shown that hydrogen bonding to the phenoxyl oxygen atom, when present, causes a reduction in spin density on O and a corresponding increase on C4. Calculated proton hyperfine coupling constants for the beta-protons show that the alpha-carbon is rotated 75-80 degrees out of the plane of the ring in PSII and Salmonella typhimurium RNR, but only 20-30 degrees in, e.g., Escherichia coli, mouse, herpes simplex, and bacteriophage T4-induced RNRs. Furthermore, based on the present calculations, we have revised the empirical parameters used in the experimental determination of the oxygen spin density in the tyrosyl radical in E. coli RNR and of the ring carbon spin densities, from measured hyperfine coupling constants.     

Hyperfine coupling of O to Mo(V) in the rapid epr signal of xanthine oxidase: Evidence for an oxygen ligand of the metal

The spectrum of the Rapid Mo(V) electron paramagnetic resonance signal from xanthine oxidase dissolved in ¹⁷O-enriched water is presented. Difference technqiues have been used to eliminate the ¹⁶O contribution. Clearly observed structure in the spectrum is attributed to moderately strong hyperfine coupling of one oxygen atom to molybdenum. Though complete interpretation of the spectrum has not been attempted, one component of A(¹⁷O) is about 1.6 mT. The possibility that the oxygen is present in a Mo---OH group, whose proton is the strongly-coupled proton of the Rapid signal, is discussed.     

Hyperfine coupling of 17O to Mo(V) in the rapid epr signal of xanthine oxidase: Evidence for an oxygen ligand of the metal

The spectrum of the Rapid Mo(V) electron paramagnetic resonance signal from xanthine oxidase dissolved in ¹⁷O-enriched water is presented. Difference technqiues have been used to eliminate the ¹⁶O contribution. Clearly observed structure in the spectrum is attributed to moderately strong hyperfine coupling of one oxygen atom to molybdenum. Though complete interpretation of the spectrum has not been attempted, one component of A(¹⁷O) is about 1.6 mT. The possibility that the oxygen is present in a MoOH group, whose proton is the strongly-coupled proton of the Rapid signal, is discussed.     

The assignment of the different infrared continuum absorbance changes observed in the 3000-1800-cm(-1) region during the bacteriorhodopsin photocycle

The bleach continuum in the 1900-1800-cm(-1) region was reported during the photocycle of bacteriorhodopsin (bR) and was assigned to the dissociation of a polarizable proton chain during the proton release step. More recently, a broad band pass filter was used and additional infrared continua have been reported: a bleach at >2700 cm(-1), a bleach in the 2500-2150-cm(-1) region, and an absorptive behavior in the 2100-1800-cm(-1) region. To fully understand the importance of the hydrogen-bonded chains in the mechanism of the proton transport in bR, a detailed study is carried out here. Comparisons are made between the time-resolved Fourier transform infrared spectroscopy experiments on wild-type bR and its E204Q mutant (which has no early proton release), and between the changes in the continua observed in thermally or photothermally heated water (using visible light-absorbing dye) and those observed during the photocycle. The results strongly suggest that, except for the weak bleach in the 1900-1800-cm(-1) region and >2500 cm(-1), there are other infrared continua observed during the bR photocycle, which are inseparable from the changes in the absorption of the solvent water molecules that are photothermally excited via the nonradiative relaxation of the photoexcited retinal chromophore. A possible structure of the hydrogen-bonded system, giving rise to the observed bleach in the 1900-1800-cm(-1) region and the role of the polarizable proton in the proton transport is discussed.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

Blood physiological resources and their role in physiological adaptation of the body to blood loss (based on the experimental data and calculations)

In white rats, loss of about 66 +/- 3% of the blood volume results in an abrupt drop of the BP and arrest of the lung respiration. Introduction of a plasma substitute in the volume equal to the lost blood resulted in an increase of the BP and restoration of rhythmical respiration in 9 out of 14 experimental rats. Haematocrit after the blood loss and subsequent administration of the substitute was about 15%. Previously obtained data show that the Ht of about 15% decreases the oxygen transport to tissues. However, an increase in the lung ventilation, a shift of the haemoglobin dissociation curve to the right, an increase of oxygen extraction from the blood, and an increase in the minute blood volume by 50-100%, allow a sufficient oxygen transport to the organism tissues to be ensured at the Ht of about 15%. Thus, the reserve of the blood respiration function of the blood remaining after a life-threatening blood loss plays an important physiological role in replenishing the volume of the lost blood with the plasma substitute and in survival of the organism.     

The contribution of the leak of protons across the mitochondrial inner membrane to standard metabolic rate

This paper presents and assesses the hypothesis that the proton leak across the mitochondrial inner membrane is an important contributor to standard metabolic rate, and that increases in the amount of mitochondrial inner membrane may be important in causing changes in proton leak and in the standard metabolic rate. The standard metabolic rate of an animal is known to be a function of body mass, phylogeny and thyroid status, and is largely attributed to the metabolically active internal organs. The total area of mitochondrial inner membrane in these organs correlates well with standard metabolic rate over a wide range of body masses in both ectotherms and endotherms. In hepatocytes isolated from rats, proton leak across the mitochondrial inner membrane accounts for about 30% of the resting oxygen consumption, and the distribution of control over respiration suggests that changes in mitochondrial inner membrane surface area will be accompanied by significant changes in the proton leak. This change in the leak will result in significant changes in resting oxygen consumption, but changes in ATP demand may also have a role to play in determining resting respiration rate. Extrapolation of these results to other tissues and other animals suggests that the hypothesis has the potential to explain a substantial proportion of the variation in standard metabolic rate with body mass, phylogeny and thyroid status. However, in most cases the quantitative contribution of proton leak compared to cellular ATP turnover has yet to be experimentally determined.     

Cellular energetics and the oxygen dependence of respiration in cardiac myocytes isolated from adult rat

The oxygen dependence of mitochondrial respiration was investigated using suspensions of mitochondria and quiescent ventricular myocytes isolated from adult rat hearts. A new optical method was used to determine oxygen concentration in the suspending media. The P50 for respiration for coupled mitochondria at a high [ATP]/[ADP].[Pi] ratio and oxidizing glutamate/malate was 0.45 +/- 0.03 microM but was increased to 0.57 +/- 0.02 microM by the addition of succinate to the substrate mixture. This value was decreased to less than 0.06 +/- 0.01 microM when the ATP/ADP.Pi ratio was decreased with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The P50 value in resting myocytes was 2.23 +/- 0.13 microM at a Vmax of 13.22 +/- 1.38 nmol of O2/g, dry weight/min. During resting conditions, the creatine phosphate/creatine and ATPfree/ADPfree ratios were high in these cells, 6.81 +/- 1.11 and 1131 +/- 185, respectively. Addition of 1 mM Ca2+ to the suspending media increased the P50 by 50% whereas respiration rose by only 10%. Respiratory rate was increased up to about 10-fold by uncoupling the cells, but the P50 increased by less than 3-fold. When these uncoupled cells were inhibited with Amytal to lower the rate of oxygen consumption to that of resting cells, the P50 fell to 1.25 +/- 0.14 microM. Diffusion models indicate that in resting myocytes, the oxygen concentration difference from sarcolemma to cell core was approximately 1.84 microM with an additional difference of about 0.27 microM attributed to the unstirred layer of media surrounding each cell. The intracellular oxygen diffusivity coefficient in myocytes was calculated to be 0.30 x 10(-5) cm2/s. The results show that the oxygen dependence of respiration is modulated by the cellular metabolic state. At near maximal levels of respiration or on recovery from hypoxic episodes, oxygen diffusion may become an important determinant of the oxygen dependence of myocardial respiration.     

Bioenergetics at extreme temperature: Thermus thermophilus ba(3)- and caa(3)-type cytochrome c oxidases

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba(3)- and caa(3)-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa(3)-oxidase subunits having been initially 'fused'. Coupled with sequence analysis, the ba(3)-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate-enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included.     

Proton exclusion by an aquaglyceroprotein: a voltage clamp study

BACKGROUND INFORMATION: In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. RESULTS: To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7x10(-14) cm(3).subunit(-1).s(-1). Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H(+) (OH(-)) to H(2)O molecules transport stoichiometry of 2x10(-9). A significant GlpF-mediated ion conductivity was also not detectable. CONCLUSIONS: The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.     

Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

Light energy conservation processes in Halobacterium halobium cells.

In Halobacterium halobium, proton pumping driven by light or by respiration generates an electrochemical potential difference across the membrane. Energy storage in this form is only transient. Cellular energy transducers competing with proton leaks stabilize this free energy as high energy phosphate bonds, electrochemical potential of other ions, and chemical potential of amino acids and possibly other chemical species. The pH changes induced by light or by respiration in cell suspensions are complicated by proton flows associated with the functioning of the cellular energy transducers. Dominant is the proton inflow coupled to the synthesis of ATP, which has been kinetically resolved. A proton-per-ATP ratio of about 3 is calculated from simultaneous measurements of photophosphorylation and the proton inflow. This value is compatible with the chemiosmotic coupling hypothesis. The time course of the light-induced changes in membrane potential indicates that light-driven pumping increases a dark preexisting potential of about 130 mV only by a small amount (20-30 mV). The complex kinetic features of the membrane potential changes do not closely follow those of the pH changes, indicating that flows of ions other than protons are involved. A qualitative model consistent with the available data is presented. A salient feature of this model is a sudden relaxation of the protonmotive force by a proton inflow through the ATPase when the preexisting protonmotive force is increased by light or respiration and reaches a critical value. The trigger could be either the proton-motive force, the pH gradient, or possibly the internal pH.     

Rapid postnatal developmental changes in the passive proton permeability of the inner membrane in rat liver mitochondria

Titration of mitochondrial respiration against the membrane potential with the inhibitor malonate has been carried out during the perinatal period in isolated rat liver mitochondria. Neonatal and adult mitochondria exhibited the characteristic "nonohmic" behavior for the proton conductance (CmH+). In contrast, fetal mitochondria exhibited an "anomalous" "ohmic" behavior for CmH+. The calculated passive proton permeability of the membrane undergoes a profound reduction during the first postnatal hour. The results reported demonstrate that the hypothesis [Pollak, J.K. & Sutton, R. (1980) Trends Biochem. Sci. 5, 23-27] of the existence of a "leaky" mitochondria in the fetal rat liver, and of its sudden neonatal change towards a state of higher energy conservation of the proton electrochemical gradient, is correct.     

Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.     

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q_D) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q_D site (MSQ_D) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with ¹H₂O/²H₂O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ_D binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in ¹H₂O and ²H₂O samples and by selective detection of the exchanged deuterons using Q-band ²H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b_D. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q_D site are discussed, in light of the unusually high thermodynamic stability of MSQ_D.     

NMR structure of the 3' stem-loop from human U4 snRNA

The NMR structure of the 3' stem-loop (3'SL) from human U4 snRNA was determined to gain insight into the structural basis for conservation of this stem-loop sequence from vertebrates. 3'SL sequences from human, rat, mouse and chicken U4 snRNA each consist of a 7 bp stem capped by a UACG tetraloop. No high resolution structure has previously been reported for a UACG tetraloop. The UACG tetraloop portion of the 3'SL was especially well defined by the NMR data, with a total of 92 NOE-derived restraints (about 15 per residue), including 48 inter-residue restraints (about 8 per residue) for the tetraloop and closing C-G base pair. Distance restraints were derived from NOESY spectra using MARDIGRAS with random error analysis. Refinement of the 20mer RNA hairpin structure was carried out using the programs DYANA and miniCarlo. In the UACG tetraloop, U and G formed a base pair stabilized by two hydrogen bonds, one between the 2'-hydroxyl proton of U and carbonyl oxygen of G, another between the imino proton of G and carbonyl oxygen O2 of U. In addition, the amino group of C formed a hydrogen bond with the phosphate oxygen of A. G adopted a syn orientation about the glycosidic bond, while the sugar puckers of A and C were either C2'-endo or flexible. The conformation of the UACG tetraloop was, overall, similar to that previously reported for UUCG tetraloops, another member of the UNCG class of tetraloops. The presence of an A, rather than a U, at the variable position, however, presents a distinct surface for interaction of the 3'SL tetraloop with either RNA or protein residues that may stabilize interactions important for active spliceosome formation. Such tertiary interactions may explain the conservation of the UACG tetraloop motif in 3'SL sequences from U4 snRNA in vertebrates.