Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO₂ ^?-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k _on for the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M^?1 s^?1 (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10^?6 M, (1.8 ± 0.2) × 10^?6 M, and (1.4 ± 0.2) × 10^?6 M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Characterization of hemoglobin from Phoronis architecta (phoronida)

• 1.1. Phoronis architecta hemoglobin is composed of four distinct hemoglobin subunits with minimum MW's of 16–17,000 or 17–19,000 daltons. All four hemoglobins are monomeric when oxygenated. Two of the monomers combine to form dimers when bound with carbon monoxide.
• 2.2. In cellulo, Phoronis architecta hemoglobin has a half-saturation (P₅₀) value of 1.3 ± 0.1 mm Hg, shows cooperative oxygen binding (Hill coefficient = 2.7 ± 0.3), and no Bohr effect from pH 6.6 to 7.9. In vitro, the hemoglobin has a P₅₀ of 0.76 ± 0.21 mm Hg but shows no cooperativity (0.90 ± 0.15 (SD)).
• 3.3. The oxygen dissociation constant (K_off) from hemoglobin is 2.7 ± 0.2 sec⁻¹, and the computed oxygen association constant (K_on) is 2.5 × 10⁶ M⁻¹ · sec⁻¹ (1.9–3.6 × 10⁶ M⁻¹ · sec⁻¹).

     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

Rapid kinetic studies of calmodulin interactions with calcium and troponin I as monitored by anthroylcholine fluorescence

Anthroylcholine was utilized as an extrinsic fluorescent probe in rapid kinetic studies of calcium dissociation from calmodulin (k_off = 10 S^?1) and the calmodulin-troponin I complex (k_off = 6 S^?1). At concentrations lower than 70 μM, the mechanism of dye binding agreed with the simple kinetic scheme in which the dye binds exclusively to the respective calcium complexes of calmodulin and calmodulin-troponin I. The sensitivity of anthroylcholine also made possible the estimation of values for the association (1.0 ± 0.8) × 10⁸$\text{M}{}^{\mathrm{?1}}$ S^?1) and dissociation rate constants (2 ± 170 S^?1) for troponin I binding to the calcium₄-calmodulin complex.     

Nitrofuran induced mutagenesis and error prone repair in Escherichia coli

The binding of cis(c)- and trans(t)-Pt(NH₃)₂Cl₂ to DNA at platinum/DNA-nucleotide ratios (R_i) of 0.1 or less has been studied by means of radioactive ^195mPt-labeled compounds. Kinetic data are consistent with the following scheme:

At 25°C and pH 5–6 in 5 mM NaClO₄, the values for the rate constants in the above scheme for the c-isomer are k₂ = 2.2 × 10^?5 sec^?1, k₇ = 0.32 (sec M)^?1, and k₈ = 143 (sec M)^?1; for the t-isomer the values are k₂ < 0.5 × 10^?5 sec^?1 and k₇ = 0.95 (sec M)^?1. Platinum-DNA adducts do not undergo detectable exchange after 3 days at 37°C, indicating the absence of a dynamic equillibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag⁺ and H⁺ titration studies are consistent with binding at guanine N(7) for R_i < 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37°C and pH 7 in 0.15 M NaCl, 6–8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.     

Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k_on, association) and reverse (k_off, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′_on = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′_on = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k_off = 0.024 s⁻¹) to 6 bp (k_off = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of k_on and k_off is measured. Interestingly, k_onincreases by >40-fold (k_on = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between [NaCl] = 25 mM and 1 M), whereas in contrast, k_offdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (K_eq) increases by ≈160, largely due to changes in the association rate, k_on. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy

We developed a new method to elucidate the binding kinetics k_on and k_off, and the dissociation constant K_D (=k_off/k_on), of protein-protein interactions without observable bound resonances of the protein of interest due to high molecular weight in a complex with a large target protein. In our method, k_on and k_off rates are calculated from the analysis of longitudinal relaxation rates of free resonances measured for multiple samples containing different concentration ratios of ¹⁵N-labeled protein and substoichiometric amounts of the target protein. The method is applicable to interactions that cannot be analyzed by relaxation dispersion spectroscopy due to slow interactions on millisecond to second timescale and/or minimal conformational (chemical shift) change upon binding. We applied the method to binding of the B1 domain of protein G (GB1) to immunoglobulin G, and derived the binding kinetics despite the absence of observable bound GB1 resonances.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

Self-exchange rates and electron transfer kinetics of horse heart ferricytochrome C with amino acid-pentacyanoferrate(II) complexes

The electron transfer reactions of horse heart cytochrome c with a series of amino acid-pentacyanoferrate(II) complexes have been studied by the stopped-flow technique, at 25°C, μ = 0.100, pH 7 (phosphate buffer). A second-order behavior was observed in the case of the Fe(CN)₅ (histidine)^3? complex, with k = 2.8 x 10⁵ M^?1 sec^?1. For the Fe(CN)₅ (alanine)^4? and Fe(CN)₅(L-glutamate)^5? complexes, only a minor deviation of the second-order behavior, close to the experimental error (k = 3.2 × 10⁵ and 1.6 x 10⁵ M^?1 sec^?1, respectively) was noted at high concentrations of the reactants (e.g., 6 × 10^?4 M). The results are in accord with recent work on the Fe(CN)₆^4?/cytochrome c system demonstrating weak association of the reactants. The calculated self-exchange rate constants including electrostatic interactions for the imidazole,L -histidine, 4-aminopyridine, glycinate, β-alaninate, andL-glutamate pentacyanoferrate(II) complexes were 3.3 × 105, 3.3 × 10⁵, 2.8 × 10⁶,4.1 × 10²,5.5 × 10², and 6.0 M^?1 sec^?1, respectively. Marcus theory calculations for the cytochrome c reactions were interpreted in terms of two nonequivalent binding sites for the complexes, with the metalloprotein self-exchange rate constants varying from 10⁴ M^?1 sec^?1 (histidine, imidazole, and 4-aminopyridine complexes) to 10⁶ M^?1 sec ^?1 (glycinate, β-alaninate, and L-glutamate complexes).     

Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from $\text{Penicillium}\text{chrysogenum}$ was studied at pH 6.18. At this sub-optimum pH, V_max was about 83 units × mg protein^?1 compared with 225 units × mg protein^?1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP⁺/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel (Renosto, F. $\text{et}\text{al.}$ J. Biol. Chem. $\text{256}$, 8616, 1981). A major effect of lowering the assay pH was to change the K_m for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which k_cat in the forward direction at pH 7.20 (ca. 375 sec^?1) is greater that k_off for the dissociation of one or more substrates. Several observations suggest that k_off for FAD is extremely small at pH 7.20.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

The designer leptin antagonist peptide Allo-aca compensates for short serum half-life with very tight binding to the receptor

The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C _max of 8.9 μg/mL at 5 min corresponds to approximately 22 % injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC₅₀ value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with k _a = 5 × 10⁵ M^?1 s^?1 and k _diss = 1.5 × 10^?4 s^?1 values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

Contributions of water transfer energy to protein‐ligand association and dissociation barriers: Watermap analysis of a series of p38α MAP kinase inhibitors

In our previous work, we proposed that desolvation and resolvation of the binding sites of proteins can serve as the slowest steps during ligand association and dissociation, respectively, and tested this hypothesis on two protein‐ligand systems with known binding kinetics behavior. In the present work, we test this hypothesis on another kinetically‐determined protein‐ligand system—that of p38α and eight Type II BIRB 796 inhibitor analogs. The k_on values among the inhibitor analogs are narrowly distributed (10⁴ ≤ k_on ≤ 10⁵ M^?1 s^?1), suggesting a common rate‐determining step, whereas the k_off values are widely distributed (10^?1 ≤ k_off ≤ 10^?6 s^?1), suggesting a spectrum of rate‐determining steps. We calculated the solvation properties of the DFG‐out protein conformation using an explicit solvent molecular dynamics simulation and thermodynamic analysis method implemented in WaterMap to predict the enthalpic and entropic costs of water transfer to and from bulk solvent incurred upon association and dissociation of each inhibitor. The results suggest that the rate‐determining step for association consists of the transfer of a common set of enthalpically favorable solvating water molecules from the binding site to bulk solvent. The rate‐determining step for inhibitor dissociation consists of the transfer of water from bulk solvent to specific binding site positions that are unfavorably solvated in the apo protein, and evacuated during ligand association. Different sets of unfavorable solvation are evacuated by each ligand, and the observed dissociation barriers are qualitatively consistent with the calculated solvation free energies of those sets.