Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species

Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes of hemeproteins. We studied the reactions between H₂O₂ and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11), and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order rate constant for HbII to decrease from 141.60 to 77.78 M⁻¹ s⁻¹, and to 2.96 M⁻¹ s⁻¹, respectively. This pH dependence is associated with the disruption of the heme–tyrosine (603 nm) protein moiety, which controls the access of the H₂O₂ to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I was evident in the UV–vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H₂O₂, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created a path much faster than 3.0×10⁻² s⁻¹ for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme–tyrosine moiety complex determined the overall reaction rate of the oxidative reaction limiting the interaction with H₂O₂ at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins.     

Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand

 Most biological substrates have distinctive sizes, shapes, and charge distributions which can be recognized specifically by proteins. In contrast, myoglobin must discriminate between the diatomic gases O₂, CO, and NO which are apolar and virtually the same size. Selectivity occurs at the level of the covalent Fe-ligand complexes, which exhibit markedly different bond strengths and electrostatic properties. By pulling a water molecule into the distal pocket, His64(E7)¹ inhibits the binding of all three ligands by a factor of ∼10 compared to that observed for protoheme-imidazole complexes in organic solvents. In the case of O₂ binding, this unfavorable effect is overcome by the formation of a strong hydrogen bond between His64(E7) and the highly polar FeO₂ complex. This favorable electrostatic interaction stabilizes the bound O₂ by a factor of ∼1000, and the net result is a 100-fold increase in overall affinity compared to model hemes or mutants with an apolar residue at position 64. Electrostatic interaction between FeCO and His64 is very weak, resulting in only a two- to three-fold stabilization of the bound state. In this case, the inhibitory effect of distal pocket water dominates, and a net fivefold reduction in K _CO is observed for the wild-type protein compared to mutants with an apolar residue at position 64. Bound NO is stabilized ∼tenfold by hydrogen bonding to His64. This favorable interaction with FeNO exactly compensates for the tenfold inhibition due to the presence of distal pocket water, and the net result is little change in K _NO when the distal histidine is replaced with apolar residues. Thus, it is the polarity of His64 which allows discrimination between the diatomic gases. Direct steric hindrance by this residue plays a minor role as judged by: (1) the independence of K _O2, K _CO, and K _NO on the size of apolar residues inserted at position 64, and (2) the observation of small decreases, not increases, in CO affinity when the mobility of the His64 side chain is increased. Val68(E11) does appear to hinder selectively the binding of CO. However, the extent is no more than a factor of 2–5, and much smaller than electrostatic stabilization of bound O₂ by the distal histidine. Received, accepted: 23 May 1997     

Chlorine redox chemistry is widespread in microbiology

Chlorine is abundant in cells and biomolecules, yet the biology of chlorine oxidation and reduction is poorly understood. Some bacteria encode the enzyme chlorite dismutase (Cld), which detoxifies chlorite (ClO₂⁻) by converting it to chloride (Cl⁻) and molecular oxygen (O₂). Cld is highly specific for chlorite and aside from low hydrogen peroxide activity has no known alternative substrate. Here, we reasoned that because chlorite is an intermediate oxidation state of chlorine, Cld can be used as a biomarker for oxidized chlorine species. Cld was abundant in metagenomes from various terrestrial habitats. About 5% of bacterial and archaeal genera contain a microorganism encoding Cld in its genome, and within some genera Cld is highly conserved. Cld has been subjected to extensive horizontal gene transfer. Genes found to have a genetic association with Cld include known genes for responding to reactive chlorine species and uncharacterized genes for transporters, regulatory elements, and putative oxidoreductases that present targets for future research. Cld was repeatedly co-located in genomes with genes for enzymes that can inadvertently reduce perchlorate (ClO₄⁻) or chlorate (ClO₃⁻), indicating that in situ (per)chlorate reduction does not only occur through specialized anaerobic respiratory metabolisms. The presence of Cld in genomes of obligate aerobes without such enzymes suggested that chlorite, like hypochlorous acid (HOCl), might be formed by oxidative processes within natural habitats. In summary, the comparative genomics of Cld has provided an atlas for a deeper understanding of chlorine oxidation and reduction reactions that are an underrecognized feature of biology.Subject terms: Soil microbiology, Water microbiology     

Spin-state equilibria and axial ligand bonding in FixL hydroxide: a resonance raman study

 Vibrational assignments for the Fe-OH unit of ferric alkaline forms of two deletion derivatives of Rhizobium meliloti FixL, FixL*, a functional O₂-sensing heme kinase, and FixLN, which contains only the heme domain, are made. Appearance of ²H- and ¹⁸O-sensitive Raman bands indicates that the heme group of FixL binds hydroxide as a distal ligand to form a six-coordinate complex. The alkaline FixLs are distributed between high- and low-spin states. The high- and low-spin bands corresponding to the ν (Fe-OH) modes occur at 479 and 539 cm^–1, respectively. Low temperature favors formation of the low-spin complex, indicative of a thermal spin-state equilibrium. The ν (Fe-OH) frequencies of FixLN and FixL* are 11 to 18 cm^–1 lower than those observed for the respective vibrations in alkaline myoglobin and hemoglobin. The weaker Fe-OH bond in the FixLs is attributed to a lack of hydrogen bonding on the distal side of the heme pocket. Received: 20 November 1997 / Accepted: 2 March 1998     

Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

Perchlorate reduction by a mixed culture in an up-flow anaerobic fixed bed reactor

Wolinella succinogenes HAP-1 is a Gram-negative microaerophile which reduces perchlorate to chloride by the proposed pathway ClO₄ to ClO₃ to ClO₂ to Cl + O₂. A cost-effective perchlorate treatment process has been established using a consortium of facultative anaerobic organisms and W. succinogenes HAP-1. The mixed anaerobic bacterial culture containing W. succinogenes HAP-1 was examined for the ability to form a biofilm capable of perchlorate reduction. An up-flow anaerobic fixed bed reactor (UAFBR) was packed with diatomaceous earth pellets and operated at residence times of 1.17 and 0.46 h to insure a viable biofilm had attached to the diatomaceous earth pellets. Reduction rates of perchlorate to chloride in the UAFBR could be maintained at 1 g of perchlorate reduced h⁻¹ L⁻¹. Studies with the same bacterial consortium in continuously stirred tank reactors (CSTR) generally reduced 0.5–0.7 g of perchlorate h⁻¹. Viable cell counts were performed periodically on the diatomaceous earth pellets and demonstrated that the W. succinogenes HAP-1 population was maintained at 28–47% of the total microbial population. Scanning electron micrographs demonstrated that the external and internal surfaces of the diatomaceous pellets were densely colonized with microbial cells of multiple cell types. This is the first report of an anaerobic mixed culture forming a biofilm capable of perchlorate reduction. Received 22 May 1997/ Accepted in revised form 07 January 1998     

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of α1β1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain β1(1–385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (β1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat β1 distal heme pocket (Ile-145 → Tyr and Ile-149 → Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe^II-O₂ complexes and is likely the first observation of a Fe^II-O₂ complex in the full-length α1β1 protein. Additionally, these data suggest that atypical sGCs stabilize O₂ binding by a hydrogen bonding network involving tyrosine and glutamine.     

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H₂O₂ to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H₂O₂. This explains the amitrole toxicity. From binding studies, the dissociation constant (K _d) for amitrole with lactoperoxidase was found to be approximately 5.5 × 10⁻⁷ M, indicating high affinity.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (k _ox ) for this complex was 8.7 × 10⁵ M⁻¹s⁻¹. The lowest value of k _ox was estimated for the complex with benzotriazole (k _ox = 3.1 × 10⁴ M⁻¹s⁻¹). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.     

Modulating carbon monoxide binding affinity and kinetics in myoglobin: the roles of the distal histidine and the heme pocket docking site

 Myoglobin has long served as a model system for understanding the relations between protein structure, dynamics, and function. Its ability to discriminate between toxic CO and vital O₂, two small ligands that are almost equivalent in size and dipole moment, has attracted much attention. To understand discrimination and reversible ligand-binding in Mb, both the bound state and the "docked" state that leads to binding need to be studied. We have reported previously the nearly linear Fe–C–O geometry of bound CO and the nearly orthogonal geometry of docked CO [Lim et al. (1995), Science 269 : 962]. With the exception of X-ray structures, a preponderance of evidence points to a nearly linear Fe–C–O geometry and calls into question the proposal that the highly conserved distal histidine forces CO to bind in a nonoptimal geometry. The differences between the bound CO structures determined using IR and X-ray methods might arise from a water molecule hydrogen bonded to the distal histidine in some of the unit cells. Discrimination by Mb is manifested not only thermodynamically but also kinetically. Time-resolved CO rebinding studies that compare Mb with microperoxidase suggest that the heme pocket docking site in Mb exerts steric control of the ligand rebinding rate, slowing the rate of CO binding by a factor of more than 10⁴. Received, accepted: 23 May 1997     

Structural and kinetic studies of imidazole binding to two members of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>6</Subscript> family reveal an important role for a conserved heme pocket residue

The amino acid at position 51 in the cytochrome c ₆ family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c ₆ Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe–S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k _obs for imidazole binding to Arabidopsis thaliana cytochrome c _6A, which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c ₆, was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s⁻¹). For the cytochrome c ₆ Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c _6A was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c ₆ family is additionally responsible for tuning the stability of the heme iron–Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997     

The TOM complex is involved in the release of superoxide anion from mitochondria

Available data indicate that superoxide anion (O₂^•− ) is released from mitochondria, but apart from VDAC (voltage dependent anion channel), the proteins involved in its transport across the mitochondrial outer membrane still remain elusive. Using mitochondria of the yeast Saccharomyces cerevisiae mutant depleted of VDAC (Δpor1 mutant) and the isogenic wild type, we studied the role of the TOM complex (translocase of the outer membrane) in the efflux of O₂^•− from the mitochondria. We found that blocking the TOM complex with the fusion protein pb₂-DHFR decreased O₂^•− release, particularly in the case of Δpor1 mitochondria. We also observed that the effect of the TOM complex blockage on O₂^•− release from mitochondria coincided with the levels of O₂^•− release as well as with levels of Tom40 expression in the mitochondria. Thus, we conclude that the TOM complex participates in O₂^•− release from mitochondria.     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.     

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture

Summary The effect of low concentrations of hydrogen peroxide (H₂O₂) (5 × 10⁻⁷−9.5 × 10⁻⁷ M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H₂O₂ (8 × 10⁻⁷ M) but H₂O₂ concentration of 7 × 10⁻⁶ M and above increased cell death. H₂O₂ stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10⁻⁷ or 1 × 10⁻⁵ M H₂O₂ was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 × 10⁻⁷ M H₂O₂ was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H₂O₂ or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10⁻⁵ M H₂O₂ which increased with increasing H₂O₂ until cell death.     

On the significance of hydrogen bonds for the discrimination between CO and O2 by myoglobin

 Quantum chemical geometry optimisations have been performed on realistic models of the active site of myoglobin using density functional methods. The energy of the hydrogen bond between the distal histidine residue and CO or O₂ has been estimated to be 8 kJ/mol and 32 kJ/mol, respectively. This 24 kJ/mol energy difference accounts for most of the discrimination between CO and O₂ by myoglobin (about 17 kJ/mol). Thus, steric effects seem to be of minor importance for this discrimination. The Fe—C and C—O vibrational frequencies of CO-myoglobin have also been studied and the results indicate that CO forms hydrogen bonds to either the distal histidine residue or a water molecule during normal conditions. We have made several attempts to optimise structures with the deprotonated nitrogen atom of histidine directed towards CO. However, all such structures lead to unfavourable interactions between the histidine and CO, and to ν_CO frequencies higher than those observed experimentally. Received: 7 July 1998 / Accepted: 26 October 1998     

Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb₁, Rb₂, Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% (w/v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l⁻¹ h⁻¹ at 1 h and was hydrolyzed completely to APPD with 364 mg l⁻¹ h⁻¹ after 5 h. Rb₁, Rb₂, Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l⁻¹ h⁻¹ at 1.2 h and were hydrolyzed completely to APPD with 300 mg l⁻¹ h⁻¹ after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.     

Oxygen uptake does not increase linearly at high power outputs during incremental exercise test in humans

A group of 12 healthy non-smoking men [mean age 22.3 (SD 1.1) years], performed an incremental exercise test. The test started at 30 W, followed by increases in power output (P) of 30 W every 3 min, until exhaustion. Blood samples were taken from an antecubital vein for determination of plasma concentration lactate [La⁻]_pl and acid-base balance variables. Below the lactate threshold (LT) defined in this study as the highest P above which a sustained increase in [La⁻]_pl was observed (at least 0.5 mmol · l⁻¹ within 3 min), the pulmonary oxygen uptake (V˙O₂) measured breath-by-breath, showed a linear relationship with P. However, at P above LT [in this study 135 (SD 30) W] there was an additional accumulating increase in V˙O₂ above that expected from the increase in P alone. The magnitude of this effect was illustrated by the difference in the final P observed at maximal oxygen uptake (V˙O_2max) during the incremental exercise test (P _max,obs at V˙O_2max) and the expected power output at V˙O_2max(P _max,exp at V˙O_2max) predicted from the linear V˙O₂-P relationship derived from the data collected below LT. The P _max,obs at V˙O_2max amounting to 270 (SD 19) W was 65.1 (SD 35) W (19%) lower (P<0.01) than the P _max,exp at V˙O_{2max .} The mean value of V˙O_2max reached at P _max,obs amounted to 3555 (SD 226) ml · min⁻¹ which was 572 (SD 269) ml · min⁻¹ higher (P<0.01) than the V˙O₂ expected at this P, calculated from the linear relationship between V˙O₂ and P derived from the data collected below LT. This fall in locomotory efficiency expressed by the additional increase in V˙O₂, amounting to 572 (SD 269) ml O₂ · min⁻¹, was accompanied by a significant increase in [La⁻]_pl amounting to 7.04 (SD 2.2) mmol · l⁻¹, a significant increase in blood hydrogen ion concentration ([H⁺]_b) to 7.4 (SD 3) nmol · l⁻¹ and a significant fall in blood bicarbonate concentration to 5.78 (SD 1.7) mmol · l⁻¹, in relation to the values measured at the P of the LT. We also correlated the individual values of the additional V˙O₂ with the increases (Δ) in variables [La⁻]_pl and Δ[H⁺]_b. The Δ values for [La⁻]_pl and Δ[H⁺]_b were expressed as the differences between values reached at the P _max,obs at V˙O_2max and the values at LT. No significant correlations between the additional V˙O₂ and Δ[La⁻]_pl on [H⁺]_b were found. In conclusion, when performing an incremental exercise test, exceeding P corresponding to LT was accompanied by a significant additional increase in V˙O₂ above that expected from the linear relationship between V˙O₂ and P occurring at lower P. However, the magnitude of the additional increase in V˙O₂ did not correlate with the magnitude of the increases in [La⁻]_pl and [H⁺]_b reached in the final stages of the incremental test. Accepted: 30 October 1997