Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-α-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-α-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (δH^? = 65 ± 2 kJ · mol^-1 and δS^? = -54 ± 7 J · mol^-1. K^-1 for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.     

Dynamics of interaction of vitamin C with some potent nitrovasodilators, S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and S-nitrosocaptopril (SNOCap), in aqueous solution

The reductive decomposition of both SNAP and SNOCap by ascorbate in aqueous solution (in the presence of EDTA) was thoroughly investigated. Nitric oxide (NO) release from the reaction occurs in an ascorbate concentration and pH dependent manner. Rates and hence NO release increased drastically with increasing pH, signifying that the most highly ionized form of ascorbate is the more reactive species. The experiments were monitored spectrophotometrically, and second-order rate constants calculated at 37 degrees C for the reduction of SNAP are k(b)=9.81+/-1.39 x 10(-3) M(-1) s(-1) and k(c)=662+/-38 M(-1) s(-1) and for SNOCap are k(b)=2.57+/-1.29 x 10(-2) M(-1) s(-1) and k(c)=49.7+/-1.3 M(-1) s(-1). k(b) and k(c) are the second-order rate constants via the ascorbate monoanion (HA-) and dianion (A2-) pathways, respectively. Activation parameters were also calculated and are DeltaHb++ =93+/-7 kJ mol(-1), DeltaSb++ =15+/-2 J K(-1) mol(-1) and DeltaHc++ =51+/-5 kJ mol(-1), DeltaSc++ =-28+/-3 J K(-1) mol(-1) with respect to the reactions involving SNAP. Those for the reaction between SNOCap and ascorbate were calculated to be DeltaHb++ =63+/-11 kJ mol(-1), DeltaSb++ =-71+/-20 J K(-1) mol(-1) and DeltaHc++ =103+/-7 kJ mol(-1), DeltaSc++ =118+/-8 J K(-1) mol(-1). The effect of Cu2+/Cu+ ions on the reductive decompositions of these S-nitrosothiols was also investigated in absence of EDTA. SNOCap exhibits relatively high stability at near physiological conditions (37 degrees C and pH 7.55) even in the presence of micromolar concentrations of Cu2+, with decomposition rate constant being 0.011 M(-1) s(-1) in comparison to SNAP which is known to be more susceptible to catalytic decomposition by Cu2+ (second-order rate constant of 20 M(-1) s(-1) at pH 7.4 and 25 degrees C). It was also observed that the reductive decomposition of SNAP is not catalyzed by alkali metal ions, however, there was an increase in rate as the ionic strength increases from 0.2 to 0.5 mol dm(-3) NaCl.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Measurement of myocardial free radical production during exercise using EPR spectroscopy

Exercise is associated with an increase in oxygen flux through the mitochondrial electron transport chain that has recently been demonstrated to increase the production of reactive oxygen species (ROS) in skeletal muscle. This study examined whether exercise also causes free radical production in the heart. We measured ROS production in seven chronically instrumented dogs during rest and treadmill exercise (6.4 km/h at 10 degrees grade; and heart rate, 204 +/- 3 beats/min) using electron paramagnetic resonance spectroscopy in conjunction with the spin trap alpha-phenyl-tert-butylnitrone (PBN) (0.14 mol/l) in blood collected from the aorta and coronary sinus (CS). To improve signal detection, the free radical adducts were deoxygenated over a nitrogen stream for 15 min and extracted with toluene. The hyperfine splitting constants of the radicals were alpha(N) = 13.7 G and alpha(H) = 1.0 G, consistent with an alkoxyl or carbon-centered radical. Resting aortic and CS PBN adduct concentrations were 6.7 and 6.3 x 10(8) arbitrary units (P = not significant). Both aortic and CS adduct concentrations increased during exercise, but there was no significant difference between the aortic and CS concentrations. Thus, in contrast to skeletal muscle, submaximal treadmill exercise did not result in detectable free radical production by the heart.     

Free radical-induced redox chemistry of platinum-containing anti-cancer drugs

Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological pH and chloride concentration conditions, hydroxyl radical reaction rate constants of (9.99 +/- 0.20) x 10(9), (8.38 +/- 0.55) x 10(9), and (6.03 +/- 0.08) x 10(9) M(-1) s(-1) at 24.0, 20.7 and 24.0 degrees C, respectively, with corresponding activation energies of 12.79 +/- 0.57, 13.88 +/- 1.14, and 14.35 +/- 0.56 kJ mol(-1) for these three reactions, were determined. These oxidations of cisplatin and transplatin to form a Pt(III) transient are significantly faster than reported previously at room temperature. The lower rate constant for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at the platinum center and the cyclobutanedicarboxylate ligand. The equivalent reductive hydrated electron reaction rate constants measured were (1.99 +/- 0.04) x 10(10) (24.0 degrees C), (1.77 +/- 0.08) x 10(10) (22.0 degrees C), and (8.92 +/- 0.06) x 10(9) M(-1) s(-1) (24.0 degrees C), with corresponding activation energies of 15.75 +/- 1.00, 19.74 +/- 1.82, and 19.99 +/- 0.34 kJ mol(-1). Again, the values determined for cisplatin and transplatin are faster than reported; however, all three values are consistent with direct reduction of the platinum center to form a Pt(I) moiety.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Acid-catalysed autoreduction of ferrylmyoblobin in aqueous solution studied by freeze quenching and ESR spectroscopy

Decay of the hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found, when studied by a combination of ESR and UV/VIS spectroscopy in aqueous solution at physiological pH, to proceed by parallel second- and first-order kinetics. At pH below 6.5 a sharp ESR signal (g = 2.003) with an increasing intensity for decreasing pH were observed in solutions frozen in liquid nitrogen, and a broad signal (g = 2.005) was seen throughout the studied pH range also in frozen solutions. The g = 2.005 signal is suggested to arise from an intermediate formed in an intramolecular rate-determining electron-transfer in ferrylmyoglobin, whereas the g = 2.003 signal is caused by a radical formed in a proton-assisted electron-transfer initiating the specific acid-catalysed autoreduction.     

Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

Volume and enthalpy profiles of CO rebinding to horse heart myoglobin

Carbon monoxide binding to myoglobin was characterized using the photothermal beam deflection method. The volume and enthalpy changes coupled to CO dissociation were found to be 9.3+/-0.8 mL x mol(-1) and 7.4+/-2.8 kcal x mol(-1), respectively. The corresponding values observed for CO rebinding have the same magnitude but opposite sign: Delta V=-8.6+/-0.9 mL x mol(-1) and Delta H=-5.8+/-2.9 kcal x mol(-1). Ligand rebinding occurs as a single conformational step with a rate constant of 5 x 10(5) M(-1) s(-1) and with activation enthalpy of 7.1+/-0.8 kcal x mol(-1) and activation entropy of -22.4+/-2.8 cal x mol(-1) K(-1). Activation parameters for the ligand binding correspond to the activation parameters previously obtained using the transient absorption methods. Hence, at room temperature the CO binding to Mb can be described as a two-state model and the observed volume contraction occurs during CO-Fe bond formation. Comparing these results with CO dissociation reactions, for which two discrete intermediates were characterized, indicates differences in mechanism by which the protein modulates ligand association and dissociation.     

Pulse radiolysis studies of beta-carotene in oxygenated DMSO solution. Formation of beta-carotene radical cation

The spectroscopic and kinetic characteristics of beta-carotene radical cation (beta-carotene(.+)) were studied by pulse radiolysis in aerated DMSO solution. The buildup of beta-carotene(.+) with k(1) = (4.8 +/- 0.2) x 10(8) dm(3) mol(-1) s(-1) [lambda(max) = 942 nm, epsilon = (1.6 +/- 0.1) x 10(4) dm(3) mol(-1) cm(-1)] results from an electron transfer from beta-carotene to DMSO(.+). The beta-carotene(.+) species decays exclusively by first-order reaction, k = (2.1 +/- 0.1) x 10(3) s(-1), probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (beta-carotene)(2)(.+). Under the experimental conditions, a small additional beta-carotene triplet-state absorption ((3)beta-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 x 10(4) s(-1)), resulting in singlet oxygen ((1)O(2)), whose reactions can also lead to additional formation of beta-carotene(.+).     

Electrochemical generation of ferrylmyoglobin during oxidation of styrene with films of DNA and a poly (ester sulfonic acid) ionomer.

The chemistry of electrochemically-driven myoglobin-catalyzed oxidation of styrene was investigated in films of DNA or Eastman AQ ionomer on optically transparent electrodes. Conversion of styrene to styrene oxide proceeded via a ferrylmyoglobin radical intermediate. Ferrylmyoglobins were clearly detected by spectroelectrochemistry in films of 1-4 mm thick. The ferrylmyoglobin radical is produced by reaction of metmyoglobin (Mb) in the films with hydrogen peroxide formed by electrochemical catalytic reduction of oxygen catalyzed by Mb. Thus, electrochemically-driven styrene oxidation with these films proceeds by a 'doubly catalytic' electrode-driven reduction-oxidation pathway. Ferrylmyoglobin formation during electrolysis of Mb-DNA films in aerobic solutions was much faster, and styrene oxidation occurred with less Mb decomposition compared to the Mb-AQ films. The better performance of Mb-DNA films is correlated with a larger fraction of electroactive Mb and better stability than for the Mb-AQ films.     

Oxidation-state-dependent reactions of cytochrome <Emphasis Type="Italic">c</Emphasis> with the trioxidocarbonate(•1−) radical: a pulse radiolysis study

The reaction of the trioxidocarbonate(*1-) radical (CO (3) (*-) , "carbonate radical anion") with cytochrome c was studied by pulse radiolysis at alkaline pH and room temperature. With iron(III) cytochrome c, CO (3) (*-) reacts with the protein moiety with rate constants of (5.1 +/- 0.6) x 10(7) M(-1) s(-1) (pH 8.4, I approximately 0.27 M) and (1.0 +/- 0.2) x 10(8) M(-1) s(-1) (pH 10, I = 0.5 M). The absorption spectrum of the haem moiety was not changed, thus, amino acid radicals produced on the protein do not reduce the haem. The pH-dependent difference in rate constants may be attributed to differences in ionization states of amino acids and to the change in the conformation of the protein. With iron(II) cytochrome c, CO (3) (*-) oxidizes the haem quantitatively, presumably via electrostatic guidance of the radical to the solvent-accessible haem edge, with a different pH dependence: at pH 8.4, the rate constant is (1.1 +/- 0.1) x 10(9) M(-1) s(-1) and, at pH 10, (7.6 +/- 0.6) x 10(8) M(-1) s(-1). We propose that CO (3) (*-) oxidizes the iron center directly, and that the lower rate observed at pH 10 is due to the different charge distribution of iron(II) cytochrome c.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Catalytic mechanism and product specificity of rubisco large subunit methyltransferase: QM/MM and MD investigations

Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have been carried out in an investigation of Rubisco large subunit methyltransferase (LSMT). It was found that the appearance of a water channel is required for the stepwise methylation by S-adenosylmethionine (AdoMet). The water channel appears in the presence of AdoMet (LSMT.Lys-NH3+.AdoMet), but is not present immediately after methyl transfer (LSMT.Lys-N(Me)H2+.AdoHcy). The water channel allows proton dissociation from both LSMT.AdoMet.Lys-NH3+ and LSMT.AdoMet.Lys-N(Me)H2+. The water channel does not appear for proton dissociation from LSMT.AdoMet.Lys-N(Me)2H+, and a third methyl transfer does not occur. By QM/MM, the calculated free energy barrier of the first methyl transfer reaction catalyzed by LSMT (Lys-NH2 + AdoMet --> Lys-N(Me)H2+ + AdoHcy) is DeltaG++ = 22.8 +/- 3.3 kcal/mol. This DeltaG++ is in remarkable agreement with the value 23.0 kcal/mol calculated from the experimental rate constant (6.2 x 10-5 s-1). The calculated DeltaG++ of the second methyl transfer reaction (AdoMet + Lys-N(Me)H --> AdoHcy + Lys-N(Me)2H+) at the QM/MM level is 20.5 +/- 3.6 kcal/mol, which is in agreement with the value 22.0 kcal/mol calculated from the experimental rate constant (2.5 x 10-4 s-1). The third methyl transfer (Lys-N(Me)2 + AdoMet --> Lys-N(Me)3+ + AdoHcy) is associated with an allowed DeltaG++ of 25.9 +/- 3.2 kcal/mol. However, this reaction does not occur because a water channel does not form to allow the proton dissociation of Lys-N(Me)2H+. Future studies will determine whether the product specificity of lysine (mono, di, and tri) methyltransferases is determined by the formation of water channels.     

In vivo production of hydroxyl radical by Enterococcus faecalis colonizing the intestinal tract using aromatic hydroxylation

Enterococcus faecalis is an intestinal commensal that produces extracellular superoxide (O(2)(*-)) through autoxidation of membrane-associated demethylmenaquinone. To assess free radical production by E. faecalis in vivo, intestinal tracts of rats were colonized using wild-type E. faecalis or a mutant strain with attenuated O(2)(*-) production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean +/- SD) showed 1.4 +/- 1.5 and 0.094 +/- 0.24 microM 5,5-dimethyl-1-pyrroline-N-oxide-hydroxyl radical adduct/gm stool for rats colonized with wild-type and mutant strains, respectively (p = .002). In vivo hydroxyl radical production was further assayed by aromatic hydroxylation using phenyl N-tert-butylnitrone (PBN) and D-phenylalanine. Hydroxylated PBN and D-phenylalanine products were recovered from stool (microM/gm colonic contents/10(9) colony forming units) and urine (microM/h/ml), respectively, and quantified using electrochemical detection. Hydroxylated (OH) PBNs and isomeric tyrosines (hydroxylated phenylalanine) were significantly increased (mean +/- SD) for rats colonized with wild-type E. faecalis (2-OH PBN, 63 +/- 58; 3-OH PBN, 63 +/- 84; ortho-tyrosine, 31 +/- 27; meta-tyrosine, 17 +/- 14) compared to the mutant strain (2-OH PBN, 2.5 +/- 7.3 (p < .001); 3-OH PBN, 3.9 +/- 12.3 (p = .01); ortho-tyrosine, 1.9 +/- 6.0 (p < .001); meta-tyrosine, 1.5 +/- 3.4 (p = .03)). Similar differences were observed following in vitro incubations of these bacteria with aromatic targets. These results confirm in vivo production of hydroxyl radical by E. faecalis colonizing the intestine, and indicate this bacterium may be a potent source of oxidative stress on the intestinal epithelium.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation Parameters for the Spontaneous and Pressure-Induced Phases of the Dissociation of Single-Ring GroEL (SR1) Chaperonin

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5-3.0 kbar. The kinetics were studied as a function of temperature in the range 15-35 degrees C. The dissociation processes at each pressure and temperature showed biphasic behavior. The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure. This dissociation was pressure independent and followed concentration-dependent first-order kinetics. The self-dissociation rates followed normal Eyring plots (In k1,obs/T vs. 1/T) from which the free energy of activation (deltaG++ = 22 +/- 0.3 kcal mol(-1)), enthalpy of activation (deltaH++ = 18 +/- 0.5 kcal mol(-1)), and entropy of activation (deltaS++ = -15 +/- 1 kcal mol(-1)) were evaluated. The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs. pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent. Activation volumes at zero pressure (V++o) and compressibility factors (beta++) for the dissociation rates at the specific temperatures were calculated. This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.     

Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle.

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The delta V of N to NClx was -30.9 +/- 1.4 mL/mol protein, whereas MG to NClx entailed a delta V of -235 +/- 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.