Free Energy Plots Research Articles

Determining binding of sulfonamide antibiotics to CTABr micelles using semi-equilibrium dialysis

Abstract Membrane filtration processes such as ultrafiltration are increasingly employed in industrial and municipal wastewater treatment. However, these filtration processes are usually poor for removing low molecular weight organic and inorganic contaminants. Micellar enhanced ultrafiltration (MEUF), where contaminants first partition into micellar phases prior to removal by ultrafiltration, has been shown to be an effective removal technique for a variety of trace contaminants in wastewater. To characterize or predict the removal efficiency of MEUF, it is important to have a quantitative measure of the extent to which a species is able to bind to a micelle as well as to develop an understanding of the dynamics of the binding interactions. Herein, we report micellar binding constants for a series of sulfonamide antibiotics (sulfadoxine, sulfathiazole, sulfamethoxazole, sulfamerazine, sulfadiazine, sulfamethazine, sulfacetamide, sulfaguanidine, and sulfanilamide) with cetyltrimethylammonium bromide (CTABr) micelles, as determined using a semi-equilibrium dialysis method. Sulfadoxine, sulfathiazole and sulfamethoxazole exhibited the strongest tendency to bind to CTABr micelles, indicating that they possessed greater potential for removal via MEUF than did sulfanilamide or sulfaguanidine, which displayed little affinity for binding with CTABr micelles. Linear free energy plots of Log KB (in CTABr) versus Log KOW and pKa2 values indicated that there was a weak correlation between binding and Log KOW and a stronger correlation with pKa2 for these compounds, likely reflecting the stronger binding of ionizable sulfonamide molecules in the anionic form by the cationic micelles.

Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures.

Standard molar Gibbs free energy and enthalpy of solvation of low polar solutes in formamide derivatives at 298 K

Novel experimental values of the limiting activity coefficients and standard molar enthalpies of solution in N-methylformamide at 298.15K are reported and used to calculate the thermodynamic functions of solvation. We compare the plots of the standard molar Gibbs free energy against the standard molar enthalpy of solvation of low polar molecules in N-methylformamide, N,N-dimethylformamide, and formamide. The solvophobic effect is responsible for the deviations of the data points from the straight line corresponding to a correlation between thermodynamic functions of solvation in aprotic solvents. It is shown that the solvophobic effects are strong in formamide solutions, significantly weaker in N-methylformamide, and very weak in N,N-dimethylformamide, which coincides with the average number of intermolecular hydrogen bonds per unit volume of these solvents. Behavior of solutions in binary solvents composed of N-methylformamide or formamide mixed with water and ethylene glycol and a possibility to tune the solvophobic effect by changing the solvent composition are also considered.

Experimental reconstruction of primary hot isotopes and characteristic properties of the fragmenting source in heavy-ion reactions near the Fermi energy

The characteristic properties of the hot nuclear matter existing at the time of fragment formation in the multifragmentation events produced in the reaction $^{64}$Zn + $^{112}$Sn at 40 MeV/nucleon are studied. A kinematical focusing method is employed to determine the multiplicities of evaporated light particles, associated with isotopically identified detected fragments. From these data the primary isotopic yield distributions are reconstructed using a Monte Carlo method. The reconstructed yield distributions are in good agreement with the primary isotope distributions obtained from AMD transport model simulations. Utilizing the reconstructed yields, power distribution, Landau free energy, characteristic properties of the emitting source are examined. The primary mass distributions exhibit a power law distribution with the critical exponent, $A^{-2.3}$, for $A \geq 15$ isotopes, but significantly deviates from that for the lighter isotopes. Landau free energy plots show no strong signature of the first order phase transition. Based on the Modified Fisher Model, the ratios of the Coulomb and symmetry energy coefficients relative to the temperature, $a_{c}/T$ and $a_{sym}/T$, are extracted as a function of A. The extracted $a_{sym}/T$ values are compared with results of the AMD simulations using Gogny interactions with different density dependencies of the symmetry energy term. The calculated $a_{sym}/T$ values show a close relation to the symmetry energy at the density at the time of the fragment formation. From this relation the density of the fragmenting source is determined to be $\rho /\rho_{0} = (0.63 \pm 0.03 )$. Using this density, the symmetry energy coefficient and the temperature of fragmenting source are determined in a self-consistent manner as $a_{sym} = (24.7 \pm 3.4) MeV$ and $T=(4.9 \pm 0.2)$ MeV.

Investigation of the nuclear phase transition using the Landau free-energy approach

Fragment yield data have been analyzed using the Landau free energy approach to investigate the critical phenomena of fragmenting quasi-projectiles formed in ${}^{64}\mathrm{Zn}\phantom{\rule{0.16em}{0ex}}+{\phantom{\rule{0.16em}{0ex}}}^{64}$Zn, ${}^{64}\mathrm{Ni}\phantom{\rule{0.16em}{0ex}}+{\phantom{\rule{0.16em}{0ex}}}^{64}$Ni, and ${}^{70}\mathrm{Zn}\phantom{\rule{0.16em}{0ex}}+{\phantom{\rule{0.16em}{0ex}}}^{70}$Zn reactions at beam energy of 35 MeV/nucleon. Pairing coefficients normalized to the temperature of the fragmenting source (${a}_{p}/T$) were extracted from yield data of $N=Z$ fragments in order to correct for odd-even effects in free energy calculations. Plots of fragment free energy ($F/T$) versus the neutron-proton asymmetry show three minima, indicating the system to be in a first-order phase transition regime. Values of fitting parameters to the Landau free energy equation as well as ${a}_{p}/T$, which are related to thermodynamic properties of the fragmenting systems, exhibit a dependence on the neutron-proton asymmetry in addition to their dependence on the excitation energy of the fragmenting source.

Properties of hydrophobic free energy found by gas–liquid transfer

The hydrophobic free energy in current use is based on transfer of alkane solutes from liquid alkanes to water, and it has been argued recently that these values are incorrect and should be based instead on gas-liquid transfer data. Hydrophobic free energy is measured here by gas-liquid transfer of hydrocarbon gases from vapor to water. The new definition reduces more than twofold the values of the apparent hydrophobic free energy. Nevertheless, the newly defined hydrophobic free energy is still the dominant factor that drives protein folding as judged by ΔCp, the change in heat capacity, found from the free energy change for heat-induced protein unfolding. The ΔCp for protein unfolding agrees with ΔCp values for solvating hydrocarbon gases and disagrees with ΔCp for breaking peptide hydrogen bonds, which has the opposite sign. The ΔCp values for the enthalpy of liquid-liquid and gas-liquid transfer are similar. The plot of free energy against the apparent solvent-exposed surface area is given for linear alkanes, but only for a single conformation, the extended conformation, of these flexible-chain molecules. The ability of the gas-liquid hydrophobic factor to predict protein stability is tested and reasonable agreement is found, using published data for the dependences on temperature of the unfolding enthalpy of ribonuclease T1 and the solvation enthalpies of the nonpolar and polar groups.

Theoretical and experimental studies of hydrogen adsorption and desorption on Ir surfaces

We report adsorption and desorption of hydrogen on planar Ir(210) and faceted Ir(210), consisting of nanoscale {311} and (110) facets, by means of temperature programmed desorption (TPD) and density functional theory (DFT) in combination with the ab initio atomistic thermodynamics approach. TPD spectra show that only one H2 peak is seen from planar Ir(210) at all coverages whereas a single H2 peak is observed at around 440 K (F1) at fractional monolayer (ML) coverage and an additional H2 peak appears at around 360 K (F2) at 1 ML coverage on faceted Ir(210), implying structure sensitivity in recombination and desorption of hydrogen on faceted Ir(210) versus planar Ir(210), but no evidence is found for size effects in recombination and desorption of hydrogen on faceted Ir(210) for average facet sizes of 5-14 nm. Calculations indicate that H prefers to bind at the two-fold short-bridge sites of the Ir surfaces. In addition, we studied the stability of the Ir surfaces in the presence of hydrogen at different H coverages through surface free energy plots as a function of the chemical potential, which is also converted to a temperature scale. Moreover, the calculations revealed the origin of the two TPD peaks of H2 from faceted Ir(210): F1 from desorption of H2 on {311} facets while F2 from desorption of H2 on (110) facets.

ROLE OF QUASIPROJECTILE ISOSPIN ASYMMETRY IN NUCLEAR FRAGMENTATION

Fragment yield data, selected with different values of the isospin asymmetry of the quasiprojectile from 78, 86 Kr +58, 64 Ni reactions at E lab = 35 MeV/nucleon, have been analyzed within the framework of Landau free energy approach. Fits to the free energy data indicated the system to be in the regime of a first-order phase transition. The plot of free energy versus isospin asymmetry of the fragments showed a systematic change with increasing isospin asymmetry of the fragmenting source. This observation demonstrated the role of quasiprojectile isospin asymmetry as an external field which governed the minima positions in the free energy plot. The position of the central minimum was found to be in close agreement with the average isospin asymmetry of the fragments calculated excluding neutrons and protons. Significant deviations from the free energy plot were observed for N = Z fragments, particularly for the lighter ones, which could be corrected after inclusion of odd–even effects in the analysis. Analysis of yield data of N = Z nuclei gave a reasonable estimate of temperature. Analysis of free energy data gated with excitation energy of the fragmenting source showed a dependence of fit parameters of Landau free energy equation on the excitation energy as well as isospin asymmetry of the source.

Theoretical investigation of experimentally determined α-εffects — The role of electronics

Recent experimental reports involving both α-nucleophiles and normal nucleophiles have reported both the presence and absence of an α-effect. In ester systems, such as dimethylmethylphosphonate (DMMP), a small α-effect is reported, but the reference point is a stationary point of the potential energy surface that must rearrange to acquire the near attack conformation (NAC) necessary for the Sn2 pathway to proceed. The second type of study involves use of highly fluorinated alkoxides as normal nucleophiles and reports no α-effect. This paper employs linear free energy plots in an investigation of electronic effects in methyl formate SN2 reactions, using high-level computations of transition states for determination of energy barriers.

Time‐dependent insulin oligomer reaction pathway prior to fibril formation: Cooling and seeding

The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

Observation of magnetocaloric effect in the LaMn1.9Fe0.1Si2 compound at low fields in the vicinity of phase transition around room temperature

Low field magnetic and magnetocaloric properties of the compound LaMn1.9Fe0.1Si2 have been investigated. Using the M-H data measured at various temperatures, we have derived the magnetic entropy change |ΔS| as a function of temperature and magnetic field, by using the Maxwell relation. The temperature dependence of the Landau coefficient has been derived by fitting the magnetization, using the Landau expansion of magnetic free energy and Arrott plots. The results show that LaMn1.9Fe0.1Si2 is ferromagnetically ordered around room temperature (285 K) and the nature of the phase transition at this temperature is found to be second order.

Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.

Magnetocaloric effect in CeCo4B compound

Abstract Polycrystalline CeCo 4 B compound was prepared by arc melting under argon atmosphere. The magnetic and magnetocaloric properties of CeCo 4 B were investigated. The magnetic measurements show that CeCo 4 B is ferrimagnetically ordered. The Curie temperature was determined to be 291 K. It was found that the maximum value of the magnetic entropy change |Δ S m | has reached the highest value of 0.361 J/(kg K) at 1 T. Using the Landau expansion of magnetic free energy and Arrott plots, it was found that the phase transition in CeCo 4 B is of second order.

Compartmentalization of Reactants in Different Regions of Sodium 1,4-Bis(2-ethylhexyl)sulfosuccinate/Heptane/Water Reverse Micelles and Its Influence on Bimolecular Electron-Transfer Kinetics

Sodium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micellar medium has been used to study the photoinduced electron-transfer (ET) reactions between some coumarin derivatives and amines, namely, aniline (AN) and N,N-dimethylaniline (DMAN) at different w(0) (w(0) = [water]/[AOT]) values, to explore the appearance of Marcus inversion and also the possible role of w(0), if any, on the Marcus correlation curves. The coumarin derivatives are found to partition between the heptane-like and the water-like phases of the reverse micelles, and their locations have been confirmed by time-resolved anisotropy measurements. Fluorescence quenching is found to depend both on the location of the coumarin molecules and on the hydrophobicity of the amine donors. Various aspects such as the effect of differential partitioning of the quenchers, the location of the probes in the two phases, the diffusion of the reactants in the micellar phase, etc. have been considered to rationalize the fluorescence quenching rates in reverse micelles. Rotational relaxation times and the diffusion parameters estimated from the anisotropy results do not show good correlation with the observed quenching rates indicating that the diffusion of reactants has no role in the quenching kinetics in reverse micelles. Marcus inversion behavior has been observed for the coumarin-amine systems in the water-like phase at a relatively high exergonicity of approximately 1.2 eV suggesting that the solvent reorganization energy contributes fully to the free energy of activation for the ET reactions in the present systems. This is in accordance with the fast solvent relaxation dynamics reported in reverse micelles. Quenching rates in the water-like phase are found to decrease or increase marginally with increasing w(0) for the coumarin-DMAN and coumarin-AN systems, respectively. This is explained on the basis of the changing solubility of these amines in the water-like phase with changing w(0) values of the reverse micelles. In the heptane-like phase, no clear inversion in the quenching rate versus free energy plot could be observed because the study could not be extended to higher exergonicity due to nonsolubility of the dye C151 in this phase. Present results, especially in the water-like phase, suggest that the confinement of reactants in micellar media can effectively remove the influence of reactant diffusion on bimolecular ET rates and thus make the systems more conducive for the observation of the Marcus inverted region.

Effects of Heme Pocket Structure and Mobility on Cytochrome c Stability

Unfolding thermodynamics of a thermophilic cytochrome c552 from Hydrogenobacter thermophilus (Ht cyt c552) and its mesophilic homologue from Pseudomonas aeruginosa (Pa cyt c551) as well as two heme pocket point mutants (Ht-Q64N and Pa-N64Q) are characterized by determination of protein stability curves (plots of unfolding free energy, DeltaG, vs T). These proteins show revealing differences in heme pocket hydrogen bonding and mobility. It previously has been shown that Asn64 in Pa cyt c551 and in Ht-Q64N interacts with the heme axial Met to fix it in a single conformation [Wen, X., and Bren, K. L. (2005) Biochemistry 44, 5225-5233]. In Ht cyt c552 and Pa-N64Q, Gln64 does not interact with the axial Met; in these variants the axial Met samples more than one conformation [Wen, X., and Bren, K. L. (2005) Inorg. Chem. 44, 8587-8593]. Here it is demonstrated that, relative to wild type, Pa-N64Q displays enhanced stability with an increase in unfolding free energy (DeltaDeltaG) of 7.1 kJ/mol and an increase in denaturation temperature (DeltaTm) of 8 degrees C. Correspondingly, Ht-Q64N is less stable than Ht cyt c552, with a DeltaDeltaG of -10 kJ/mol and a DeltaTm of -10 degrees C. Analysis of unfolding thermodynamics indicates that the net changes in stability resulting from the position 64 mutations are primarily attributable to entropic factors. For Pa-N64Q (Ht-Q64N) it is proposed that the favorable release (unfavorable burial) of residue 64 is the dominant factor impacting stability. The mobility of the axial Met also is proposed to contribute. These results provide a specific illustration of how amino acid side chain mobility and burial or release contribute to protein stability.

Entropy–enthalpy compensation in solution properties of solutes at infinite dilution in nonpolar polymers

AbstractThe knowledge of the Flory–Huggins interaction parameter, χ, between organic liquids and polymers is very important in the study of their miscibility. From the temperature dependency of χ the enthalpy and residual entropy of solution can be determined. In this study literature data of thirty‐two solutes, ranging from alkanes to alcohols, at infinite dilution in isotatic polypropylene, poly(ethyl ethylene), and poly(dimethylsiloxane), were tested and linear entropy–enthalpy compensation was observed. The plot of residual free energy versus enthalpy of solution was also linear, with a lower correlation coefficient than the entropy–enthalpy plot. The range of enthalpy of solution was wider than those of the size corrected free energy of solution. In nonpolar solvents the enthalpy of solution reflected largely the interaction within the solute liquid state, and showed a linear trend with respect to solute cohesive energy density for n‐alcohols. The wide range of enthalpy of solution suggests the use of a two‐dimensional solubility parameter model to correlate the enthalpy of solution. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 1241–1247, 2007

The Stability Curve of Hen Egg White Lysozyme

The physiological stability curve--a plot of the free energy of unfolding versus temperature--is calculated for hen egg white lysozyme from a combination of extrapolated unfolding thermodynamic data from reversible conditions and isothermal titrations with guanidine hydrochloride. The shape of the curve suggests the existence of only one folded conformation.

The fragmentation of protonated tyrosine and iodotyrosines: The effect of substituents on the losses of NH 3 and of H 2O and CO

The gas-phase dissociation chemistries of protonated 3-iodo- l-tyrosine, 3,5-diiodo- l-tyrosine and 3,3′,5,5′-tetraiodo-thyronine (thyroxine) have been examined using a combination of tandem mass spectrometry and density functional theory (DFT) calculations. It was found that, at low collision energy, all protonated tyrosines exhibit common fragmentation pathways, including the competitive eliminations of NH 3 and the concomitant loss of H 2O and CO, but there are significant differences in relative abundances, depending on the combined electron-donating abilities of the substituents in the phenyl ring. The ions initially formed by loss of NH 3 are phenonium ions, but subsequent fragmentation is most easily understood in terms of the isomeric benzyl cation structures. These [ M + H − NH 3] + ions fragment at relatively low collision energies, mainly by loss of ketene; by contrast, the [ M + H − H 2O − CO] + ions are more stable towards dissociation. At higher collision energies, losses of one, two and even three iodine atoms were observed. DFT calculations (at the B3LYP/DZVP level of theory) were performed on protonated 3-iodotyrosine to compare the reaction profiles for the fragmentation mechanisms. The iodo-substituent in the 3-position is weakly electron-withdrawing and this results in a barrier (27.5 kcal/mol at 0 K) that is slightly higher than that for protonated tyrosine (26.8 kcal/mol). The phenoxy group PhO– is a weaker electron-donor than HO– and protonated 3,5-diiodo-4-phenoxytyrosine has an even higher barrier (31.1 kcal/mol) to NH 3 loss than protonated 3,5-diiodotyrosine (28.8 kcal/mol). Linear free energy plots for Δ H 0 ° ‡ and Δ G 298 ° ‡ against σ + for the four protonated tyrosine derivatives show good correlations. More importantly, as the products of the dissociation are higher in energy than the transition states to their formation, the plots of Δ H 0 ° and Δ G 298 ° for the overall reaction for NH 3 loss also correlate very well with σ + (correlation coefficients of 0.99 and 0.98, respectively). The positive slopes of these Hammett plots show that the barriers to the loss of NH 3 by the neighboring-group mechanism are increased by the presence of electron-withdrawing groups in the phenyl rings.

GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.

The Reactions of Nitrosyl Complexes with Cysteine

The reaction kinetics of a set of ruthenium nitrosyl complexes, {(X)5MNO}n, containing different coligands X (polypyridines, NH3, EDTA, pz, and py) with cysteine (excess conditions), were studied by UV-vis spectrophotometry, using stopped-flow techniques, at an appropriate pH, in the range 3-10, and T = 25 degrees C. The selection of coligands afforded a redox-potential range from -0.3 to +0.5 V (vs Ag/AgCl) for the NO+/NO bound couples. Two intermediates were detected. The first one, I1, appears in the range 410-470 nm for the different complexes and is proposed to be a 1:1 adduct, with the S atom of the cysteinate nucleophile bound to the N atom of nitrosyl. The adduct formation step of I1 is an equilibrium, and the kinetic rate constants for the formation and dissociation of the corresponding adducts were determined by studying the cysteine-concentration dependence of the formation rates. The second intermediate, I2, was detected through the decay of I1, with a maximum absorbance at ca. 380 nm. From similar kinetic results and analyses, we propose that a second cysteinate adds to I1 to form I2. By plotting ln k1(RS-) and ln k2(RS-) for the first and second adduct formation steps, respectively, against the redox potentials of the NO+/NO couples, linear free energy plots are obtained, as previously observed with OH- as a nucleophile. The addition rates for both processes increase with the nitrosyl redox potentials, and this reflects a more positive charge at the electrophilic N atom. In a third step, the I2 adducts decay to form the corresponding Ru-aqua complexes, with the release of N2O and formation of cystine, implying a two-electron process for the overall nitrosyl reduction. This is in contrast with the behavior of nitroprusside ([Fe(CN)5NO]2-; NP), which always yields the one-electron reduction product, [Fe(CN)5NO]3-, either under substoichiometric or in excess-cysteine conditions.