Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.     

Interactions between carnosine and zinc and copper: implications for neuromodulation and neuroprotection

This review examines interactions in the mammalian central nervous system (CNS) between carnosine and the endogenous transition metals zinc and copper. Although the relationship between these substances may be applicable to other brain regions, the focus is on the olfactory system where these substances may have special significance. Carnosine is not only highly concentrated in the olfactory system, but it is also contained in neurons (in contrast to glia cells in most of the brain) and has many features of a neurotransmitter. Whereas the function of carnosine in the CNS is not well understood, we review evidence that suggests that it may act as both a neuromodulator and a neuroprotective agent. Although zinc and/or copper are found in many neuronal pathways in the brain, the concentrations of zinc and copper in the olfactory bulb (the target of afferent input from sensory neurons in the nose) are among the highest in the CNS. Included in the multitude of physiological roles that zinc and copper play in the CNS is modulation of neuronal excitability. However, zinc and copper also have been implicated in a variety of neurologic conditions including Alzheimer's disease, Parkinson's disease, stroke, and seizures. Here we review the modulatory effects that carnosine can have on zinc and copper's abilities to influence neuronal excitability and to exert neurotoxic effects in the olfactory system. Other aspects of carnosine in the CNS are reviewed elsewhere in this issue.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.     

Molecular level investigations of the inter- and intramolecular interactions of pH-responsive artificial triblock proteins

Intelligent materials that can undergo physical gelation in response to environmental stimuli have potential impacts in the bioengineering and biomedical fields where the entrapment of cellular or molecular species is desired. Here, we utilize atomic force microscopy (AFM) to perform molecular level investigations of designer artificial proteins that undergo physical gelation. These are engineered as triblock copolymers with independent interchain binding and solvent retention functions, namely, two terminal leucine zipper-like peptide sequences and a central alanylglycine rich sequence, respectively. AFM force measurements between probes and surfaces functionalized with molecules of this triblock protein revealed adhesive interactions that increased in average force and frequency as the pH was lowered from pH 11.2 to 7.4 to 4.5, reflecting an increase in the numbers of interacting molecular strands. In bulk solution, lowering the pH results in a viscous liquid to gel transition. The modular design of the triblock protein was also exploited for single molecule force spectroscopy investigations, which revealed altered intramolecular interactions in response to changes in pH. An increased understanding of the inter- and intramolecular forces involved in biomolecule driven gelation processes is not only of great fundamental interest in the study of the biomolecular systems involved but may also prove key in enabling the rational design of new generations of intelligent hydrogel systems.     

Determinants of histamine recognition: implications for the design of antihistamines

Towards understanding how histamine, a vital neurotransmitter, can perform multiple physiological tasks, an analysis of the different proteins that bind histamine is reported here. Their structural comparison reveals conformational rigidity of histamine. Yet, flexibility in the modes of histamine binding has been observed, which appears to suit specific biological roles of the proteins. These results will be helpful in developing specific antihistamines and also in understanding the pharmacological and toxicological profiles of existing antihistamines.     

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.     

Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes. Evidence for the participation of other residues.

One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma- and pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120 x s(-1) to 1000 x s(-1) at pH 7.0 and room temperature [extremes are hen and turkey egg white (120 x s(-1)) and human milk (1000 x s(-1))]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.     

Protective effects of carnosine on dehydroascorbate-induced structural alteration and opacity of lens crystallins: important implications of carnosine pleiotropic functions to combat cataractogenesis

The high level of dehydroascorbic acid (DHA) in the lenticular tissue is an important risk factor for the development of age-related cataracts. In this study, the effects of DHA on structure and function of lens crystallins were studied in the presence of carnosine using gel mobility shift assay, different spectroscopic techniques, and lens culture analysis. The DHA-induced unfolding and aggregation of lens proteins were largely prevented by this endogenous dipeptide. The ability of carnosine to preserve native protein structure upon exposure to DHA suggests the essential role of this dipeptide in prevention of the senile cataract development. Although the DHA-modified α-crystallin was characterized by altered chaperone activity, functionality of this protein was significantly restored in the presence of carnosine. The increased proteolytic instability of DHA-modified lens proteins was also attenuated in the presence of carnosine. Furthermore, the assessment of lens culture suggested that DHA can induce significant lens opacity which can be prevented by carnosine. These observations can be explained by the pleiotropic functions of this endogenous and pharmaceutical compound, notably by its anti-glycation and anti-aggregation properties. In summary, our study suggests that carnosine may have therapeutic potential in preventing senile cataracts linked with the increased lenticular DHA generation, particularly under pathological conditions associated with the oxidative stress.     

Interfacial and lipid transfer properties of human phospholipid transfer protein: implications for the transfer mechanism of phospholipids

In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was approximately 6 nm and that of HDL3 approximately 8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-PLTP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer.     

Norovirus-host interaction: implications for disease control and prevention

Noroviruses (NVs) are a major cause of acute gastroenteritis epidemics in both developing and developed countries and affect people of all ages. Three main human histo-blood group antigens (HBGAs) - the ABO, Lewis and secretor families - are involved in NV recognition and eight strain-specific receptor-binding patterns in two major binding groups have been described. The receptor-binding interface is located at the outermost surface of the P domain of the viral capsid. Each interface contains two major binding sites and each site interacts with a carbohydrate side-chain of the HBGAs via multiple hydrogen bonds. Soluble HBGAs in human milk are able to block binding of NV to HBGA receptors, suggesting a potential decoy receptor for the protection of infants from NV infection. Phylogenetic analysis has revealed limited genetic relatedness among NVs with similar receptor-binding patterns. This review summarises and discusses recent advances and highlights implications for future studies in the control and prevention of NV gastroenteritis.     

Comparative chlorine and temperature tolerance of the oyster Crassostrea madrasensis: implications for cooling system fouling

Crassostrea madrasensis is an important fouling oyster in tropical industrial cooling water systems. C. madrasensis individuals attach to surfaces by cementing one of their two valves to the substratum. Therefore, oyster fouling creates more problems than mussel fouling in the cooling conduits of power stations, because unlike the latter, the shell of the former remains attached to the substratum even after the death of the animal. However, there are no published reports on the tolerance of this species to chlorination and heat treatment. The mortality pattern and physiological behaviour (oxygen consumption and filtration rate) of three size groups (13 mm, 44 mm and 64 mm mean shell length) of C. madrasensis were studied at different residual chlorine concentrations (0.25, 0.5, 0.75, 1, 2, 3 to 5 mg 1-1) and temperatures (30 degrees C to 45 degrees C). The effect of shell size (= age) on C. madrasensis mortality in the presence of chlorine and taking into account temperature was significant, with the largest size group oysters showing highest resistance. At 1 mg l-1 residual chlorine, the 13 mm and 64 mm size group oysters, took 504 h (21 d) and 744 h (31 d), respectively to reach 100% mortality. At 39 degrees C, the 13 mm size group oysters took 218 min to reach 100% mortality, whereas the 64 mm size group oysters took 325 min. The oxygen consumption and filtration rate of C. madrasensis showed progressive reduction with increasing residual chlorine concentrations. However, the filtration rate and oxygen consumption responses of C. madrasensis were not significantly different between 30 degrees C (control) and 37.5 degrees C. There was a sharp decrease in the filtration rate and oxygen consumption at 38.5 degrees C. A comparison of the present mortality data with previous reports on other bivalves suggests that the chlorine tolerance of C. madrasensis lies in between that of Perna viridis and Perna perna, while its temperature tolerance is significantly higher than that of the other two bivalve species. However, in power station heat exchangers, where simultaneous chlorine and thermal stresses are existent, C. madrasensis may have an edge over other common foulants, because of its high temperature tolerance.     

Solvent isotope effects in intramolecular catalysis: Acyl transfer reactions of 4-nitrophenyl 5-nitrosalicylate in aqueous tris buffer

A kinetic study of the reactions of 4-nitrophenyl 5-nitrosalicylate in aqueous Tris buffer at 25°C has revealed three kinetically significant reactions: Tris aminolysis of the un-ionized substrate, Tris aminolysis of the ionized substrate, and spontaneous hydrolysis of the ionized substrate. Solvent isotope effects have been measured for all three reactions. Mechanisms are discussed, and the conclusion is reached that intramolecular catalysis is operating in only the spontaneous hydrolysis.     

Rendez-vous in the oviduct: implications for superovulation and embryo transfer

The meeting between the maternal and paternal gametes is dependant upon a number of complicated processes. On the maternal side it involves maturation of the oocytes under the influence on both peripheral and follicular endocrine factors. Deviations in the normal pattern of maturation will lead to ovulation of inferior oocytes. On the paternal side the transport of spermatozoa in the female genital tract following mating is an area of great importance. The establishment of the sperm reservoir in the isthmus is dependant upon a number of factors (intracellular calcium concentrations, oligo-saccharides, change in estradiol: progesterone ratio in the afferent blood supply). Alterations of the normal micro-environment may disturb both binding, release and transport as a whole. The process of fertilization occurs in the ampullar region of the oviduct and it involves several well tuned steps: binding to the zona pellucida where the acrosome reaction takes place, penetration, fusion between the oolemma and the sperm plasma membrane, activation with the release of the cortical granules, decondensation of the sperm chromatin, pronucleus formation and finally syngamy where the two pronuclei fuses. The egg will experience the first cleavage shortly thereafter. Superovulation may disturb a number of these processes including oocyte maturation (arrest at MI) and sperm and zygote transport in the oviduct caused by the deviant endocrine environment, thus leading to a higher incidence of lack of fertilization and poor embryos quality.     

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.