Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Domain separation precedes global unfolding of rhodanese

The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.     

Characterization of rhodanese-tetracyanonickelate. An active site complex that slows sulfur-free rhodanese conversion to inert conformers

The structure of the rhodanese-tetracyanonickelate (E X Ni(CN)2-4) complex has been characterized here in spectral and physical studies using urea as a structural perturbant. UV difference absorption, sedimentation velocity ultracentrifugation, fluorescence, and circular dichroism data show no significant conformational differences between sulfur-free rhodanese (E) and the E X Ni(CN)2-4 complex. The urea-induced enzyme structural transition curves were noncoincident when different structural parameters were monitored. For E, the urea concentrations giving half-maximal change (Cm) were: Cm = 3.0 M for activity measurement; Cm = 2.8 M for protein intrinsic fluorescence intensity; Cm = 4.3 M for ellipticity at 220 nm; and Cm = 3.3 M for wavelength of fluorescence emission maximum. For the E X Ni(CN)2-4 complex, Cm was shifted to a higher urea concentration relative to that found for E when activity (Cm = 3.6 M) and native protein fluorescence (Cm = 3.6 M) were the measured parameters but not when the wavelength of the emission maximum and ellipticity were monitored. Furthermore, urea-induced rhodanese structural changes were time-dependent and Ni(CN)2-4 binding on E slowed enzyme inactivation that is associated with structural relaxations. These findings, that Ni(CN)2-4 affects structural relaxations in rhodanese, are of particular interest in light of the recent suggestion that the E X Ni(CN)2-4 complex mimics a normally inaccessible intermediate in catalysis.     

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.     

On the influence of fructose-6-phosphate, ammonium, ATP and AMP on the conformation of the phosphofructokinase from rabbit muscle.

By means of circular dichroism measurements in the range from 210-240 nm and 250-300 nm it is shown that conformational changes of the phosphofructokinase from rabbit muscle correlate with the action of effectors on the enzyme. The postulated active and inactive states differ in the secondary structure. Addition of activators causes a conformational change which is interpreted as the transition of the inactive into the active state. At 210-240 nm no reversion could be observed but at 250-300 nm there are some indications for at least partial reversion.     

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmstr?m (Malmstr?m, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.     

Evidence for dynamically determined conformational states in rhodanese catalysis

Physical and kinetic studies have been used to explore hysteretic effects that are observed in rhodanese catalysis at pH 5 and also at neutral pH when the ionic strength of the medium is high. Experiments that involve observation of changes in intrinsic protein fluorescence of the enzyme and kinetic investigation of its interactions with product thiocyanate anion at pH 5 have implicated enzyme isomerization as the cause of hysteresis. Taken all together, the data indicate that the conformations of enzyme forms in the catalytic cycle are dynamically determined, depending on the relative rates of conformational relaxation and catalysis as influenced by the concentrations of substrates and products.     

pH dependence of the tryptophan fluorescence in cytochrome c oxidase: further evidence for a redox-linked conformational change associated with CuA

When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Participation of H+ in the Ca2(+)-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase

The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.     

Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5′-phosphate-dependent enzyme catalyzing CO₂ release from the α-carboxyl group of l-glutamate to yield γ-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5′-phosphate-Lys²⁷⁶ Schiff base with the distal nitrogen of His⁴⁶⁵, the penultimate residue in the GadB sequence. Herein we show that His⁴⁶⁵ has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His⁴⁶⁵ contributes with n ≈ 2.5 to the overall cooperativity of the system. The residual cooperativity (n ≈ 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His⁴⁶⁵, dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His⁴⁶⁵, a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His⁴⁶⁵ mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.     

Additive stimulatory effect of extracellular calcium and potassium on non-transferrin ferric iron uptake by HeLa and K562 cells

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability of the N-domain. While N-domain does not affect the stability of full-length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.     

Low concentrations of guanidinium chloride expose apolar surfaces and cause differential perturbation in catalytic intermediates of rhodanese

The conformations of sulfur-free and sulfur-containing rhodanese were followed with and without the detergent lauryl maltoside after guanidinium chloride (GdmCl) addition to 5 M to study the apparent irreversibility of denaturation. Without lauryl maltoside, sulfur-containing rhodanese denatured in a transition giving, at approximately 2.3 M GdmCl, 50% of the total denaturation induced change observed by activity, CD, or intrinsic fluorescence. Sulfur-free rhodanese gave more complex behavior by intrinsic fluorescence and CD. CD showed loss of secondary structure in a broad, complex, and apparently biphasic transition extending from 0.5 to 3 M GdmCl. The interpretation of the transition was complicated by time-dependent aggregation due to noncovalent interactions. Results with the apolar fluorescence probe 2-anilinonaphthalene-8-sulfonic acid, implicated apolar exposure in aggregation. Sulfhydryl reactivity indicated that low GdmCl concentrations induced intermediates affecting the active site conformation. Lauryl maltoside prevented aggregation with no effect on activity or any conformational parameter of native enzyme. Transitions induced by GdmCl were still observed and consistent with several phases. Even in lauryl maltoside, an increase in apolar exposure was detected by 2-anilinonaphthalene-8-sulfonic acid, and by protein adsorption to octyl-Sepharose well below the major unfolding transitions. These results are interpreted with a model in which apolar interdomain interactions are disrupted, thereby increasing active site accessibility, before the intradomain interactions.     

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.     

Stable intermediates can be trapped during the reversible refolding of urea-denatured rhodanese

The enzyme rhodanese (EC 2.8.1.1) could be reversibly refolded from urea in the presence of lauryl maltoside, beta-mercaptoethanol, and sodium thiosulfate. The unfolding/folding transition monitored using intrinsic fluorescence was resolved into two two-state transitions with midpoints at 3.6 and 5.0 M urea. The analysis assumed an intermediate with an emission maximum at 345 nm. Monitoring anisotropy of intrinsic fluorescence also gave an asymmetric transition. Activity followed one two-state transition centered at 3.6 M urea with no major change of secondary structure. Without thiosulfate or mercaptoethanol, there was one two-state transition at 5.0 M urea giving a species, in dilute urea, with a fluorescence maximum at 345 nm. This intermediate slowly relaxed toward 335 nm (t1/2 = 85 min) if only thiosulfate was absent but without regaining activity. Subsequent addition of thiosulfate led to a first-order recovery of activity (t1/2 = 75 min). Thus, a possible folding intermediate can be trapped which displays increased access of water and solutes to its fluorescent tryptophans. This intermediate conformer, which is flexible, has considerable secondary structure, is inactive, has exposed hydrophobic surfaces, and requires specific reducing conditions to regain full activity. Refolding probably involves an initial, rapid, hydrophobic collapse with acquisition of secondary structure to form the intermediate, followed by slower adjustment to the native global conformation. Final reactivation requires reduction localized at the active site.     

Thermally perturbed rhodanese can be protected from inactivation by self-association

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30–40°C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 µg/ml is inactivated at 40°C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 µg/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30–40°C. Light scattering kinetics at 40°C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions: at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Gluteraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40°C but not at 23°;C. Precipitates formed at 40°C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

Active site modifications quench intrinsic fluorescence of rhodanese by different mechanisms

Beef liver rhodanese can be modified covalently at the active site (Cys-247) either reversibly or irreversibly by sulfur, selenium, iodoacetate, and hydrogen peroxide. Each derivative shows an intrinsic fluorescence lower than that of the free enzyme. The reaction of rhodanese with iodoacetate or hydrogen peroxide is time-dependent and accompanied by enzyme inactivation, by the loss of one or two sulfhydryl groups, respectively, by quenching and bathochromic shift of fluorescence, and by an absorbance perturbation in the near UV. The latter findings are indicative for a displacement of some tryptophyl side chains from hydrophobic to hydrophilic environment. The fluorescence decays of the various rhodanese derivatives can be fitted by a double-exponential function with two lifetimes: a shorter one of 1-1.7 ns and a longer one of 2.8-4.6 ns. The S-loaded and Se-loaded rhodanese samples have proportionally shorter lifetimes and lower quantum yields. No such proportionality was observed for the iodoacetate-treated and for the hydrogen peroxide treated enzyme. These findings indicate that two different quenching mechanisms are operating in rhodanese derivatives, a long-range energy transfer from tryptophan to persulfide (or sulfoselenide) group and a static quenching accompanying a conformational change of the protein after modification of the active site.