Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites.

Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37 degrees C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The delta H of binding is strongly temperature-dependent for both sites, arising from a large negative delta Cp of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 A, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).     

Isolation and characterization of two peroxidases from Cucumis sativus

Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.     

The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein

The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.     

Ferricytochrome c encapsulated in silica nanoparticles: structural stability and functional properties

Using a modified sol-gel technique, we have succeeded in encapsulating ferric cytochrome c in silica nanoparticles obtained from hydrolysis and polycondensation of tetramethylorthosilicate. Particles dimensions have been determined with dynamic light scattering; this technique yields an hydrodynamic radius of about 100 nm, each nanoparticle containing about 10(2)-10(3) proteins. If stored in the cold at low ionic strength, nanoparticles are stable for more than one week, even if a slow radius increase with time is observed. CD measurements show that encapsulated proteins exhibit substantially increased stability against guanidinium hydrochloride induced denaturation. Reduction kinetics of encapsulated ferric cytochrome c by sodium dithionite, measured with standard stopped flow techniques, are slower by a factor of ten with respect to those measured in solution. Analogous experiments with myoglobin suggest that this slowing down is due to the diffusion time of dithionite within the silica matrix. Indeed, if a smaller ligand like CO is used, the intrinsic kinetic properties of encapsulated proteins are found to be unaltered even in the millisecond time range. The reported data show that our nanoparticles are extremely useful both for basic research, to study the stability and functions of encapsulated proteins, and for their potential biotechnological applications.     

Exchangeability of N termini in the ligand-gated porins of Escherichia coli

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.     

Heterogeneity of the cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein.

The cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein, exists in multiple molecular forms. Most molecules are found as two chain (MR approximately 220 000 per chain) disulfide-bridged dimeric units but several minor components of smaller size have also been identified; based upon their migration rates in dodecyl sulfate gel electrophoretic experiments, the smaller molecules characterized in this study range in molecular size from 235 000 to 146 000. The component of molecular weight 235 000 apparently represents a two chain disulfide-bridged derivative of larger parent molecules (one chain of 220 000 plus a smaller remnant), whereas smaller CIg components appear to be single chain proteins. These observations plus electrophoretic analyses of samples of plasmic digests of CIg indicate that the interchain disulfide bridging in the two chain molecule is located in a segment within approx. 175 residues of the NH2- or COOH-terminus.     

Ordered complexes of cytochrome c fragments. Kinetics of formation of the reduced (ferrous) forms

The kinetics of formation of noncovalently bound ferrous complexes derived from fragments of horse heart cytochrome c have been investigated. When the reactions are initiated by combining ferrous heme fragments with an appropriate apofragment, in the presence of 50 mM imidazole, second order rate processes are observed with rate constants essentially the same as those reported with ferric heme fragments (Parr, G. R., and Taniuchi, H. (1979) J. Biol. Chem. 254, 4836-4842). An additional, probably consecutive, kinetic process is also demonstrated. If imidazole is not present in the reaction buffer, the kinetic profiles are dramatically altered. While this is partially due to aggregation (dimerization) of the ferrous heme fragments, it can nevertheless be demonstrated that the complementation reactions with apofragments are much faster than those observed with the corresponding ferric heme fragments (in the absence of imidazole). These results reflect the effect of the oxidation state of the heme iron on the folding mechanism and, thus, the manifold nature of protein folding pathways. The rate of reduction of productive ferric complexes by sodium ascorbate was investigated and biphasic reactions were found in all cases. The data indicate an equilibrium between two forms of the ferric complexes. The results of an experiment in which the complementation of ferric (1-25)H and (23-104) was carried out in the presence of sodium ascorbate indicate that the intermediate complex (Parr, G. R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 8914-8918) is not reducible by ascorbate. Thus, the increase in oxidation-reduction potential occurring on formation of the productive complex from the unbound heme fragment occurs at a late stage of the overall reaction, possibly coinciding with ligation of methionine 80 to the heme iron.     

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism

Ca²⁺-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca²⁺ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca²⁺ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext- (deleted aa 8-408); kin- (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC₅₀ of about 140 nM. A previous study has shown that under identical conditions the kin- and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.     

An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase.

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.     

Proton NMR spectroscopy of cytochrome c-554 from Alcaligenes faecalis

Cytochrome c-554 from the bacterium Alcaligenes faecalis (ATCC 8750) is a respiratory electron-transport protein homologous to other members of the cytochrome c family. Its structure has been studied by 1H NMR spectroscopy in both the ferric and ferrous states. The ferric spectrum is characterized by downfield hyperfine-shifted heme methyl resonances at 46.25, 43.60, 38.40, and 36.73 ppm (25 degrees C, pH 7.1). Chemical shifts of these resonances change with temperature opposite to expectations derived from Curie's law. The pH behavior of the hyperfine-shifted resonances titrates with a pK of 6.3 that has been interpreted as due to ionization of a heme propionate. In the ferrous state, heme methyl, meso, and thioether bridge resonances have been observed and assigned. All aromatic proteins have been assigned according to the side chain of origin, and the structural environment about the sole tryptophan residue has been examined. The electron-transfer rate between ferric and ferrous forms has been estimated to be on the order of 3 X 10(8) M-1 s-1, which is the largest such self-exchange rate yet observed for a cytochrome.     

Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120

Heme is an iron-containing cofactor that aside from serving as the active group of essential proteins is a key element in the control of many molecular and cellular processes. In prokaryotes, the family of Fur (ferric uptake regulator) proteins governs processes essential for the survival of microorganims such as the iron homeostasis. We show that purified recombinant FurA from Anabaena sp. PCC 7120 interacts strongly with heme in the micromolar range and this interaction affects the in vitro ability of FurA to bind DNA, inhibiting that process in a concentration-dependent fashion. Our results provide the first evidence of the possible involvement of heme in the regulatory function of cyanobacterial Fur.     

Differences in protein-protein association networks for lung adenocarcinoma: A retrospective study

Various methods to determine the connectivity scores between groups of proteins associated with lung adenocarcinoma are examined. Proteins act together to perform a wide range of functions within biological processes. Hence, identification of key proteins and their interactions within protein networks can provide invaluable information on disease mechanisms. Differential network analysis provides a means of identifying differences in the interactions among proteins between two networks. We use connectivity scores based on the method of partial least squares to quantify the strength of the interactions between each pair of proteins. These scores are then used to perform permutation-based statistical tests. This examines if there are significant differences between the network connectivity scores for individual proteins or classes of proteins. The expression data from a study on lung adenocarcinoma is used in this study. Connectivity scores are computed for a group of 109 subjects who were in the complete remission and as well as for a group of 51 subjects whose cancer had progressed. The distributions of the connectivity scores are similar for the two networks yet subtle but statistically significant differences have been identified and their impact discussed.     

Proton NMR spectroscopy of sulfmyoglobin

The 1H NMR spectra of ferrous sulfmyoglobin, metsulfmyoglobin, and ferric cyanosulfmyoglobin were obtained at 300 MHz. Hyperfine-shifted resonances are observed in the case of metsulfmyoglobin and ferric cyanosulfmyoglobin that have line widths and cover a chemical shift range that are comparable to the corresponding forms of normal myoglobin. Two methyl resonances are observed in the spectrum of ferric cyanosulfmyoglobin at 44.19 and 25.48 ppm (25 degrees C, pH 8.3) that have been assigned to heme methyls at the 8- and 5-positions on the basis of pH titration effects homologous to the corresponding methyl resonances in ferric cyanomyoglobin. Examination of aromatic region resonances and the pH titration profiles of histidine resonances lead to the conclusion that the overall conformation of sulfmyoglobin was highly homologous to that of normal myoglobin and afforded assignments of histidine residues of the former. The most likely position for the addition of a sulfur atom to the heme of sulfmyoglobin is pyrrole ring A, with ring B a possible, but less likely, alternative.     

The mechanism of ion polarisation along DNA double helices

The orientation curves of short DNA fragments induced by electric field pulses are measured with high time resolution and analysed by efficient deconvolution techniques. A small, but clearly detectable delay of the 'on-field' orientation can be described accurately by the superposition of two exponential processes with opposite amplitudes. The time constant of the faster process is around 10 ns and the slower one in the range 50-1000 ns depending upon the electric field strength and chain length of the DNA fragment. The relation between amplitudes and time constants observed for each curve corresponds exactly to that expected for a convolution of two processes, where the first process is without optical response and becomes detectable only via the optical response of the second process. These results indicate that the first process reflects the polarisation of the ion atmosphere required for the second process of the orientation. Measurements at different ion concentrations c demonstrate that the reciprocal time constant of the fast process is a linear function of c and thus is consistent with an association reaction. The association rate constant evaluated from this dependence according to a simple bimolecular reaction model is 8 X 10(9) M-1 s-1 for a 95 base-pair fragment and is consistent with binding of Na+ to the helix, a reaction close to the limit of diffusion control. The association rate constant is almost independent of the electric field strength E, while the dissociation rate constant k- strongly increases with E, indicating dissociation of ions at high E values. The data suggest a linear correlation between log(k-) and E2 corresponding to a reaction driven by a dipole change. The apparent dipole change evaluated from this dependence is in the order of magnitude estimated for an elementary step of ion dissociation at one end of the helices. The combined results obtained from the polarisation and the orientation mechanism can be explained by dissociation of surprisingly few counterions biased towards one end of the helices. The experimental data obtained for a 76 base-pair fragment are analogous to those for the 95 base-pair fragment, whereas the 'slow' ion polarisation has not been detected for a fragment with 27 base-pairs. This result together with those obtained for the longer fragments at low field strengths indicate that there is a fast polarisation mechanism without 'ion dissociation' at low chain lengths and for low electric field strengths. This mechanism is replaced at high chain lengths and/or high electric field strengths by the ion dissociation mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Folding/unfolding kinetics of mutant forms of iso-1-cytochrome c with replacement of proline-71

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. To assign the effects of perturbations at position 71 to steps in the process of protein folding, the kinetic properties of the folding/unfolding reactions of normal protein and the three mutant forms are compared. At pH 6.0, 20 degrees C, fluorescence-detected folding/unfolding kinetics are monitored below, within, and above the equilibrium transition zone by using stopped-flow mixing to perform guanidine hydrochloride concentration jumps. Three kinetic phases are detected for each of the four proteins. The fastest of these phases (tau 3) differs in rate for the wild type and mutant proteins. The remaining kinetic phases (tau 1 and tau 2) have similar rates for all four proteins over the entire range of folding/unfolding conditions. The guanidine hydrochloride dependence of the relative amplitudes of the kinetic phases is complex and is sensitive to the nature of the substituent at position 71: each of the four proteins shows differences in the fraction of folding/unfolding associated with the two fastest rate processes. The results suggest that it is the location of the mutation in the primary structure rather than the nature of the substituent that determines which kinetic step (or steps) is changed in rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein conformation from electron spin relaxation data.

Electron spin relaxation data from five ferric proteins are analyzed in terms of the fractal model of protein structures. Details of this model are presented. The results lead to a characterization of protein structures by a single parameter, the fractal dimension, d. This structural parameter is shown to determine the temperature dependence of the Raman electron spin relaxation rate, which varies as T3 + 2d. Computations of d are made using x-ray data for 17 proteins. The results range from d = 1.76 for lysozyme to d = 1.34 for ferredoxin. These values are compared with values of d obtained from the present electron spin relaxation data on five ferric proteins. Typical results are d = 1.34 +/- 0.06 from relaxation data and 1.34 +/- 0.05 from x-ray data for ferredoxin; d = 1.67 +/- 0.03 from relaxation data and 1.66 +/- 0.05 from x-ray data for ferricytochrome c. The data thus support the theoretical model. Applications of this spin resonance technique to the study of changes in protein conformation are discussed.     

Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans

A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed. In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate. The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process. The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments. The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C. A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions. The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions. Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation. The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation.     

Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate

Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.