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C Bonaventura

Publications and source records attributed to C Bonaventura.

At least 19 recordsLinked to original sources
PubMed2000

Concentration-dependent effects of anions on the anaerobic oxidation of hemoglobin and myoglobin.

The redox potentials of hemoglobin and myoglobin and the shapes of their anaerobic oxidation curves are sensitive indicators of globin alterations surrounding the active site. This report documents concentration-dependent effects of anions on the ease of anaerobic oxidation of representative hemoglobins and myoglobins. Hemoglobin (Hb) oxidation curves reflect the cooperative transition from the T state of deoxyHb to the more readily oxidized R-like conformation of metHb. Shifts in the oxidation curves for Hb A(0) as Cl(-) concentrations are increased to 0.2 m at pH 7.1 indicate preferential anion binding to the T state and destabilization of the R-like state of metHb, leading to reduced cooperativity in the oxidation process. A dramatic reversal of trend occurs above 0.2 m Cl(-) as anions bind to lower affinity sites and shift the conformational equilibrium toward the R state. This pattern has been observed for various hemoglobins with a variety of small anions. Steric rather than electronic effects are invoked to explain the fact that no comparable reversal of oxygen affinity is observed under identical conditions. Evidence is presented to show that increases in hydrophilicity in the distal heme pocket can decrease oxygen affinity via steric hindrance effects while increasing the ease of anaerobic oxidation.

Animals↗
PubMed1999

Disulfide bond reduction: A powerful, chemical probe for the study of structure-function relationships in the hemocyanins.

The copper-containing hemocyanins are a class of oxygen-transport proteins whose structures differ in arthropods and molluscs. Crystal structure analyses and amino acid sequence comparisons show that disulfide bonding is a common feature in both arthropod and mollusc hemocyanins. Reduction of the disulfide bonds of a representative set of arthropod and mollusc hemocyanins results in complete loss of their oxygen-binding capacities. Thus, retention of the disulfide bonds is essential to the functional integrity of the oxygen-binding sites in the subunits of this class of oxygen carriers, despite the very different architectures of the arthropod and mollusc molecules. Depending upon the specific hemocyanin, partial to virtually complete restoration of the oxygen-binding capacity occurs when the disulfide-bond reductant is removed by dialysis. The rate at which the functional, active-site geometry is lost and the extent to which it can be restored varies markedly with hemocyanin type, aggregation state, and experimental conditions. Consequently, a comparison of these differences provides a simple, but powerful, way to probe internal and environmental factors that govern physiologically important structure-function relationships in this entire class of oxygen-transport proteins.

Animals↗
PubMed1999

Effects of S-nitrosation on oxygen binding by normal and sickle cell hemoglobin.

S-Nitrosated hemoglobin (SNO-Hb) is of interest because of the allosteric control of NO delivery from SNO-Hb made possible by the conformational differences between the R- and T-states of Hb. To better understand SNO-Hb, the oxygen binding properties of S-nitrosated forms of normal and sickle cell Hb were investigated. Spectral assays and electrospray ionization mass spectrometry were used to quantify the degree of S-nitrosation. Hb A(0) and unpolymerized Hb S exhibit similar shifts toward their R-state conformations in response to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2, 3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb that accommodate NO adduction. A cycle of deoxygenation/reoxygenation does not cause loss of NO or appreciable heme oxidation. There is, however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and its associated vasoactivity depend on the abundance of thiols and other factors that influence transnitrosation reactions. The increased oxygen affinity and R-state character that result from S-nitrosation of Hb S would be expected to decrease its polymerization and thereby lessen the associated symptoms of sickle cell disease.

Allosteric Regulation↗
PubMed1999

Altered ligand rebinding kinetics due to distal-side effects in hemoglobin chico (Lysbeta66(E10) --> thr).

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues.

Binding Sites↗
PubMed1999

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide.

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.

Adult↗
PubMed1999

Spectroelectrochemistry of heme proteins: effects of active-site heterogeneity on Nernst plots.

In order to detect and model the effect of functional chain heterogeneity on Nernst plots for heme proteins, we examined the redox properties of various myoglobins (Mbs) and their mixtures using an improved spectroelectrochemical method. Specific redox responses and formal half potentials (E1/2) were obtained for Aplysia, horse, and sperm whale Mbs, as well as 1:1 mixtures of Mbs consisting of Aplysia/sperm whale, sperm whale/horse, and horse/Aplysia. Linear Nernst plots with slopes near unity were observed for horse, sperm whale, and Aplysia Mbs, with E1/2 values of 14, 19, and 96 mV (vs. NHE) respectively, consistent with previous reports using indirect methods. The Nernst plot responses for mixtures of some of these Mbs allowed us to evaluate and model the non-Nernstian behavior that results from intrinsically different values of E1/2 and from incomplete spectral overlap. The data demonstrate that increasing the E1/2 differences between the components of a Mb mixture increases the changes in shape of the resulting Nernst plots, the dominant effect being a decrease in the observed Nernst coefficient (nNernst). Comparison of Nernst plots for redox data with Hill plots for O2 binding data shows that the redox process is more affected by the structural differences in the distal heme pockets of the Mbs studied than is O2 binding. Similar effects of chain heterogeneity may give rise to disproportionate reductions in the slopes of Nernst and Hill plots for hemoglobins (Hbs). This possibility is discussed in relation to Hbs investigated for redox and O2 binding activity in our laboratories where we find nNernst to be commonly less than nHill over a range of experimental conditions.

Algorithms↗
PubMed1998

Active-site disruption in native Limulus hemocyanin and its subunits by disulfide-bond reductants: a chemical probe for the study of structure-function relationships in the hemocyanins.

The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins.

Animals↗
PubMed1998

Conformational fluctuations in deoxy hemoglobin revealed as a major contributor to anionic modulation of function through studies of the oxygenation and oxidation of hemoglobins A0 and Deer Lodge beta2(NA2)His --> Arg.

Organisms rely on regulation at the molecular level, such as the allosteric regulation of hemoglobin (Hb) function by anions, to meet challenges presented by changing environmental and physiological conditions. A comparison of the effects of anions on oxygenation, oxidation, and sulfhydryl reactivity of Hb leads us to suggest that a large and significant part of the shift in oxygen affinity brought about by anion binding occurs as a result of increased conformational rigidity of the T state of deoxy Hb. As conformational rigidity increases, it becomes increasingly difficult for subunits in the deoxygenated T-state tetramer to assume higher oxygen affinity forms (T', T", T"'...) with less steric hindrance. The oxygen affinity reflects the average of the rapidly equilibrating conformations within the T state and is correspondingly decreased when anion levels are increased. The initial stage of the oxidation of Hb is relatively insensitive to steric alterations and thus reflects, primarily, the electronic aspects of the quaternary (T, T', T", T"'...) <--> equilibrium. We show that the reactivity of the sterically obscured sulfhydryl of beta93 Cys in deoxy Hb is much greater in chloride-free buffers than in buffers with added chloride. Anion-induced decreases in the extent and frequency of conformational fluctuations of subunits within the T-quaternary state thus reduce sulfhydryl reactivity as well as oxygen affinity. This parallel in anionic control of function allowed us to test, and disprove, the possibility that uncompensated positive charges in the central cavity of Hb Deer Lodge increase the frequency and extent of conformational fluctuations in its deoxy structure. This Hb variant exhibits increased anion sensitivity, increased oxygen affinity, and increased ease of oxidation, but without increased reactivity of its sulfhydryl groups, indicating that active-site alterations in deoxy Hb Deer Lodge are primarily electronic and not associated with increased conformational fluctuations within its T state. The restoration of normal properties in Hb Deer Lodge by addition of anions reinforces our conclusion that anionic control can be exerted through both steric and electronic alterations. The anionic control of fluctuations within the T state of Hb illustrates an important principle of macromolecular structure-function relationships: that functional regulation can be achieved by alterations in conformational rigidity.

Anions↗
PubMed1997

Healthy environments for healthy people: bioremediation today and tomorrow.

Increases in environmental contamination lead to a progressive deterioration of environmental quality. This condition challenges our global society to find effective measures of remediation to reverse the negative conditions that severely threaten human and environmental health. We discuss the progress being made toward this goal through application of bioremediation techniques. Bioremediation generally utilizes microbes (bacteria, fungi, yeast, and algae), although higher plants are used in some applications. New bioremediation approaches are emerging based on advances in molecular biology and process engineering. Bioremediation continues to be the favored approach for processing biological wastes and avoiding microbial pathogenesis. Bioremediation may also play an increasing role in concentrating metals and radioactive materials to avoid toxicity or to recover metals for reuse. Microbes can biodegrade organic chemicals; purposeful enhancement of this natural process can aid in pollutant degradation and waste-site cleanup operations. Recently developed rapid-screening assays can identify organisms capable of degrading specific wastes and new gene-probe methods can ascertain their abundance at specific sites. New tools and techniques for use of bioremediation in situ, in biofilters, and in bioreactors are contributing to the rapid growth of this field. Bioremediation has already proven itself to be a cost-effective and beneficial addition to chemical and physical methods of managing wastes and environmental pollutants. We anticipate that it will play an increasingly important role as a result of new and emerging techniques and processes.

Animals↗
PubMed1997

Stroma-free human hemoglobin A decreases R3230Ac rat mammary adenocarcinoma blood flow and oxygen partial pressure.

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.

Adenocarcinoma↗
PubMed1996

Nitrate binding to Limulus polyphemus subunit type II hemocyanin and its functional implications.

The horseshoe crab, Limulus polyphemus, employs hemocyanin as an oxygen carrier in its hemolymph. This hemocyanin displays cooperative oxygen binding and heterotropic allosteric regulation by protons, chloride ions and divalent cations. Here, we report the crystal structure of Limulus polyphemus subunit type II hemocyanin with a nitrate ion bound in the interface of its first and second domains. Interestingly, the nitrate-binding site coincides with the binding site for the allosteric effector chloride. Oxygen-binding data indeed indicate that nitrate, like chloride, reduces the oxygen affinity of this hemocyanin. The observed binding of two distinct anions to a single site suggests that several other anions may also bind at this site. This opens the intriguing possibility that bicarbonate, which is structurally similar to nitrate and closely linked to respiration, can act as an allosteric effector that lowers the oxygen affinity. Such an effect could be another factor in the repertoire of allosteric regulators of this hemocyanin; however, the physiological implications will be a challenge to decipher, since there exists a complex interplay of effects between bicarbonate, chloride, pH and divalent cations.

Animals↗
PubMed1996

S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control.

A dynamic cycle exists in which haemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated, and the NO group is released during arterial-venous transit. The vasoactivity of S-nitrosohaemoglobin is promoted by the erythrocytic export of S-nitrosothiols. These findings highlight newly discovered allosteric and electronic properties of haemoglobin that appear to be involved in the control of blood pressure and which may facilitate efficient delivery of oxygen to tissues. The role of S-nitrosohaemoglobin in the transduction of NO-related activities may have therapeutic applications.

Allosteric Regulation↗
PubMed1996

Keyhole limpet hemocyanin: structural and functional characterization of two different subunits and multimers.

Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

Amino Acid Sequence↗
PubMed1996

A carbon monoxide irreducible form of cytochrome c oxidase and other unusual properties of the "monomeric" shark enzyme.

Contrary to previous reports, the functional and spectral properties of "monomeric" shark cytochrome c oxidases are not entirely similar to those of the "dimeric" beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a "g = 12" signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.

Animals↗
PubMed1996

Structural basis for the root effect in haemoglobin.

The remarkable ability of root effect haemoglobins to pump oxygen against high O2 gradients results from extreme, acid-induced reductions in O2 affinity and cooperativity. The long-sought mechanism for the root effect, revealed by the 2 angstrom crystal structure of the ligand-bound haemoglobin from Leiostomus xanthurus at pH 7.5, unexpectedly involves modulation of the R-state. Key residues strategically assemble positive-charge clusters across the allosteric beta1 beta2-interface in the R-state. At low pH, protonation of the beta N terminus and His 147(HC3)beta within these clusters is postulated to destabilize the R-state and promote the acid-triggered, allosteric R-->T switch with concomitant O2 release. Surprisingly, a set of residues specific to root effect haemoglobins recruit additional residues, conserved among most haemoglobins, to produce the root effect.

Allosteric Regulation↗
PubMed1995

Interaction of alpha-lipoic acid enantiomers and homologues with the enzyme components of the mammalian pyruvate dehydrogenase complex.

Lipoic acid (alpha-lipoic acid, thioctic acid) is applied as a therapeutic agent in various diseases accompanied by polyneuropathia such as diabetes mellitus. The stereoselectivity and specificity of lipoic acid for the pyruvate dehydrogenase complex and its component enzymes from different sources has been studied. The dihydrolipoamide dehydrogenase component from pig heart has a clear preference for R-lipoic acid, a substrate which reacts 24 times faster than the S-enantiomer. Selectivity is more at the stage of the catalytic reaction than of binding. The Michaelis constants of both enantiomers are comparable (Km = 3.7 and 5.5 mM for R- and S-lipoic acid, respectively) and the S-enantiomer inhibits the R-lipoic acid dependent reaction with an inhibition constant similar to its Michaelis constant. When three lipoic acid homologues were tested, RS-1,2-dithiolane-3-caproic acid was one carbon atom longer than lipoic acid, while RS-bisnorlipoic acid and RS-tetranorlipoic acid were two and four carbon atoms shorter, respectively. All are poor substrates but bind to and inhibit the enzyme with an affinity similar to that of S-lipoic acid. No essential differences with respect to its reaction with lipoic acid enantiomers and homologues exist between free and complex-bound dihydrolipoamide dehydrogenase. Dihydrolipoamide dehydrogenase from human renal carcinoma has a higher Michaelis constant for R-lipoic acid (Km = 18 mM) and does not accept the S-enantiomer as a substrate. Both enantiomers of lipoic acid are inhibitors of the overall reaction of the bovine pyruvate dehydrogenase complex, but stimulate the respective enzyme complexes from rat as well as from Escherichia coli. The S-enantiomer is the stronger inhibitor, the R-enantiomer the better activator. The two enantiomers have no influence on the partial reaction of the bovine pyruvate dehydrogenase component, but do inhibit this enzyme component from rat kidney. The implications of these results are discussed.

Animals↗
PubMed1995

The site of the redox-linked proton pump in eukaryotic cytochrome c oxidases.

The electronic spectra of fully oxidized derivatives of some cytochrome c oxidase preparations are distinctly pH dependent. In general, the observed spectral shifts are greater in the case of pulsed derivatives compared to resting preparations and also, greater for preparations of the enzyme from shark skeletal muscle compared to beef heart. The low temperature near-infrared magnetic circular dichroism spectrum of the fully oxidized shark enzyme is not pH dependent in the experimental range, indicating the sensitivity of the visible region electronic spectrum to variation in pH to be due principally to changes at the heme a3-CuB chromophore. The results are discussed in relation to proposed mechanisms of proton translocation in cytochrome c oxidase.

Animals↗