pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

An Ascorbate-induced Absorbance Change in Chloroplasts from Violaxanthin De-epoxidation

A new ascorbate-induced chloroplast absorbance change which has the characteristics of a carotenoid shift is described. The absorbance change was light-dependent at pH 7 but not at pH 5. The difference spectra for the light and dark changes were similar, showing a large absorbance peak at 505 nanometers, smaller peaks near 468 and 437 nanometers, and a sharp valley around 483 nanometers. The absorbance change is assigned to violaxanthin de-epoxidation because various conditions affected the absorbance change and violaxanthin de-epoxidation similarly, and the difference spectrum resembled the spectrum of zeaxanthin minus violaxanthin in organic solvent.     

The reaction of hemin with H2O2

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.     

Determination of the dead time of a stopped-flow fluorometer

This investigation was carried out to develop a convenient alternative method for examining the performance and determining the dead time of a stopped-flow fluorometer. We examined the kinetics for the formation of the fluorescent Mg2+-8-hydroxyquinoline chelate in aqueous solutions. The reversible association of the Mg2+ ion with 8-hydroxyquinoline is a second-order process whose on and off rate constants are dependent on pH. We estimated that the Mg2+ ion chelate has a fluorescence quantum yield of 0.02 in aqueous solutions. Using this reaction we measured the dead time of a stopped-flow fluorometer at different pH values. Measurements of the dead time were found to be reproducible and accurate. The Mg2+-8-hydroxyquinoline reaction fulfills the requirements for a convenient test reaction for dead time measurement of stopped-flow fluorometers. Although the usefulness of the reaction is primarily to determine the dead times of stopped-flow instruments operating in the fluorescence mode, the reaction can also be used for testing an instrument operating in the absorbance mode.     

Optical measurement of aqueous potassium concentration by a hydrophobic indicator in lipid vesicles

An assay was developed for K+ in aqueous solution at neutral pH. The method was based on the change in optical absorbance of the hydrophobic indicator 7-(n-decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphthl++ +-1-ol (MEDPIN) in phospholipid vesicles. Formation of a ternary complex between a valinomycin-K+ pair and the anionic form of MEDPIN in the bilayer resulted in an absorption band at 584 nm. K+ concentration was determined by monitoring the MEDPIN absorbance at 584 nm and MEDPIN quenching of lissamine rhodamine B sulfonylphosphatidylethanolamine (L-RhB-PE) fluorescence by an energy-transfer mechanism. Both the fluorescence intensity and lifetime of L-RhB-PE decreased by more than 25% upon addition of 50 mM K+. Kinetic studies using stopped-flow photometry showed a single-exponential reaction of MEDPIN and valinomycin in vesicles with aqueous K+ (maximum rate 1.7 s-1) that was dependent upon [valinomycin] and [K+]. The lipid surface charge was shown to influence the ratio of anionic to neutral MEDPIN at constant pH, and to alter the sensitivity of MEDPIN absorbance to aqueous [K+]. A 1:20 neutral/negative lipid mole ratio was optimal for K+ detection at pH 7.4. Spectroscopic and kinetic data suggest that the optical response of MEDPIN to K+ involves the formation of a ternary complex between K+, valinomycin and MEDPIN.     

Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.     

A stopped-flow study of the reaction between mercuric reductase and NADPH

The reaction of the FAD-containing enzyme, mercuric reductase, with NADPH has been studied by stopped-flow kinetic methods at 25 degrees C, pH 7.3. The results suggest that the reaction involves at least three steps. The first step is very rapid and is essentially complete within the dead time of the stopped-flow apparatus. This step is associated with decreasing absorbances at 340 nm (NADPH) and 455 nm (FAD), whereas there is little formation of the absorbance at 530 nm characterizing 2-electron-reduced enzyme subunits (EH2). The second step involves an increase of the absorbance at 530 nm. The third step results in an increase of the intensity of the long-wavelength band and a change of its shape. A second equivalent of NADPH per FAD is required for this step. It is proposed that the product is an EH2-NADPH complex. In addition to these rapid steps, slow absorbance changes are also observed.     

"Schizophrenic" micellization associated with coil-to-helix transitions based on polypeptide hybrid double hydrophilic rod-coil diblock copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.     

Effect of differences in optical properties of intermediate oxygenated species of hemoglobin A0 on Adair constant determination

Careful evaluation of the so-called isosbestic properties of oxygenated and deoxygenated hemoglobin spectra demonstrates that the spectral changes are not strictly linear with respect to the degree of saturation. In order to quantify the extent of nonlinearity, optical measurements of O2 binding to human hemoglobin were made at different wavelengths in the Soret region approaching the presumed isosbestic point. The results indicate that the extinction coefficient of intermediate oxygenated hemoglobin is 1% less than that of the fully oxygenated hemoglobin, with a resulting 3% (+/- 0.15%) nonlinearity effect on measurements taken at the peak of the oxygenated hemoglobin spectrum (414 nm). The lack of isosbestic conditions allows one to investigate the functional properties of the oxygenated intermediates directly. The small difference in the absorbance of different oxygenated species has practically no influence on the determination of Adair constants at wavelengths removed from the critical isosbestic region.     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid-induced unfolding of myoglobin triggered by a laser pH jump method.

Using 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) as a photoactivatable caged proton, we could induce complete acid unfolding of myoglobin with a single nanosecond laser pulse. This was possible because of the high ( approximately mM) concentration of protons released by the photolabile compound. The ability of the compound to produce a large pH jump arises because the other photoproducts (2-nitrosoacetophenone and sulfate ion) do not buffer the released protons. The complete time course of the unfolding kinetics, spanning a range from milliseconds to several seconds, could be accurately reproduced by monitoring absorbance changes in the visible spectrum at 633 nm.     

Rhodopsin in nanodiscs has native membrane-like photointermediates

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) ? Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.     

Spectral and kinetic studies on the interaction between oxyhaemoglobin and peroxynitrite: a comparison with hydrogen peroxide.

Interaction between oxyhaemoglobin and peroxynitrite was studied using stopped-flow rapid-scan spectrophotometry. The influence of pH, peroxynitrite concentration and temperature on the pseudo-first-order rate constants was studied and the activation energy calculated. The kinetic curve for the oxyhaemoglobin-peroxynitrite reaction showed that a fast reaction occurred in the initial seconds, followed by a slow process of decrease in absorbance. The biphasic reaction kinetics of oxyhaemoglobin with peroxynitrite or hydrogen peroxide demonstrated the existence of an intermediary species. For the first time a rapid-scan stopped-flow spectrophotometry study is presented, yielding spectral and kinetic data of the reaction.     

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid-scan stopped-flow spectrophotometry at 5 degrees C in the pH range 5.1-9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH-independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching (k = 145 s-1) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4, becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol-flavin C-4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electron-reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step (k = 48 s-1 at pH 9.5) is thought to include the formation of an NADP+ complex of two-electron-reduced enzyme, while the final step (k = 6.3 s-1 at pH 9.5) results in the above-mentioned NADPH complex with two-electron-reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.     

Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin. Optical and NMR spectroscopy characterization

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme-HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10-500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme-HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme-HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.     

pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.

On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid- and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 in equilibrium with D1 in equilibrium with D2 in equilibrium with D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow "double-jump" technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme.(ABSTRACT TRUNCATED AT 250 WORDS)