pH-induced Structural Transitions Research Articles

Computational Investigations of a pH-Induced Structural Transition in a CTAB Solution with Toluic Acid.

In this work, molecular dynamics simulations were performed to study the pH-induced structural transitions for a CTAB/p-toluic acid solution. Spherical and cylindrical micelles were obtained for aqueous surfactants at pH 2 and 7, respectively, which agrees well with the experimental observations. The structural properties of two different micelles were analyzed through the density distributions of components and the molecular orientations of CTA+ and toluic acid inside the micelles. It was found that the bonding interactions between CTA+ and toluic in spherical and cylindrical micelles are very different. Almost all the ionized toluic acid (PTA−) in the solution at pH 7 was solubilized into the micelles, and it was located in the CTA+ headgroups region. Additionally, the bonding between surfactant CTA+ and PTA− was very tight due to the electrostatic interactions. The PTA− that penetrated into the micelles effectively screened the electrostatic repulsion among the cationic headgroups, which is considered to be crucial for maintaining the cylindrical micellar shape. As the pH decreased, the carboxyl groups were protonated. The hydration ability of neutral carboxyl groups weakened, resulting in deeper penetration into the micelles. Meanwhile, their bonding interactions with surfactant headgroups also weakened. Accompanied by the strengthen of electrostatic repulsion among the positive headgroups, the cylindrical micelle was broken into spherical micelles. Our work provided an atomic-level insights into the mechanism of pH-induced structural transitions of a CTAB/p-toluic solution, which is expected to be useful for further understanding the aggregate behavior of mixed cationic surfactants and aromatic acids.

PH-Induced structural transitions in whey protein isolate and ultrasonically solubilized Persian gum mixture

The present work evidently reports that ultrasonic depolymerization strongly enhanced complex coacervation between Persian gum (PG) and whey protein isolate (WPI). PG was sonicated at 60 °C, operating frequency of 20 kHz and nominal power output of 800 W for various times followed by mixing with WPI. Acid-induced interaction between the two biopolymers was studied by turbidity, light scattering, zeta potential and viscosity measurements over a wide pH range. Sonication of intact PG (IPG) for 10 min considerably reduced the molecular weight from 4.12 × 106 to 0.76 × 106 g/mol. Besides, ultrasonic fragmentation of water insoluble fraction of PG drove protein containing chains into the soluble phase. Sonicated PG (SPG) was shown to be more flexible with higher number of carboxyl groups available for electrostatic interaction with WPI, such that the complete neutralization did not occur even at protein to polysaccharide ratio of 50: 1. Additionally, scattered light intensity and viscosity measurements revealed two maxima in the pH ranges of 4.4–4.85 and 3.27–4.0, being highly intense for the gum sonicated for 10 min and longer. Considering the pH-behavior of WPI components, the former peak was related to interpolymer complex formation between β-lactoglobulin and long chain fraction of SPG, while the latter was attributed to intrapolymer association of α-lactalbumin with the short chain oligosaccharides arising from ultrasonic degradation of PG.

PH-induced structural transition during complexation and precipitation of sodium caseinate and ε-Poly-l-lysine

ε-Polylysine (EPL) is a food-grade antimicrobial peptide that forms complexes with proteins. Such complexes are potential carriers for targeted delivery of agents. To elucidate the formation of such complexes, the pH-induced phase transition of EPL and sodium caseinate (SC) complexes were characterized in terms of ionic strengths (I) and EPL/SC weight ratios (r). Electrostatic nanocomplexes (e.g. r = 2−3, I = 2 mM) were formed near the isoelectric point of SC using turbidimetry, dynamic light scattering, and ζ-potential measurements. Phase analyses revealed that the formation of nanocomplexes primarily depends on the I, and saturated binding was recorded above r = 2−2. Electrostatic potential modelling of EPL was employed to describe the interaction affinity. A three-dimensional phase boundary curve was established which divided the complexation into a nano-scale and phase separation. Atomic force microscopy images confirmed that nanocomplexes were spherical particles with uniform shapes. Morphologic examination using optical and scanning electron microscopy and Fourier transform infrared spectroscopy revealed that the nanocomplexes formed “sponge-like” precipitates at larger length scales. This work reveals the possible mechanism that drives the complexation of sodium caseinate and ε-Poly-l-lysine. This is expected to guide the construction of tailor-made protein complexes in industrial applications.

Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change

pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin α (ProTα) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil–α-helix transition of ProTα and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid–liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles.

Stress-induced acidification may contribute to formation of unusual structures in C9orf72-repeats

BackgroundExpansion of the C9orf72 hexanucleotide repeat (GGGGCC)n·(GGCCCC)n is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both strands of the C9orf72 repeat have been shown to form unusual DNA and RNA structures that are thought to be involved in mutagenesis and/or pathogenesis. We previously showed that the C-rich DNA strands from the C9orf72 repeat can form four-stranded quadruplexes at neutral pH. The cytosine residues become protonated under slightly acidic pH (pH 4.5–6.2), facilitating the formation of intercalated i-motif structures. MethodsUsing CD spectroscopy, UV melting, and gel electrophoresis, we demonstrate a pH-induced structural transition of the C-rich DNA strand of the C9orf72 repeat at pHs reported to exist in living cells under stress, including during neurodegeneration and cancer. ResultsWe show that the repeats with lengths of 4, 6, and 8 units, form intercalated quadruplex i-motifs at low pH (pH < 5) and monomolecular hairpins and monomolecular quadruplexes under neutral-basic conditions (pH ≥ 8). Furthermore, we show that the human replication protein A (RPA) binds to the G-rich and C-rich DNA strands under acidic conditions, suggesting that it can bind to i-motif structures. ConclusionsIn the proper sequence context, i-motif structures can form at pH values found in some cells in vivo. General significanceDNA conformational plasticity exists over broad range of solution conditions.

Intrinsically Disordered Proteins PH-Induced Structural Transitions in Overcrowded Milieu

Intrinsically Disordered Proteins PH-Induced Structural Transitions in Overcrowded Milieu

Molecular Dynamics Simulation of the pH-Induced Structural Transitions in CTAB/NaSal Solution.

We performed molecular dynamics simulations to study the pH-induced structural transitions for aqueous mixtures of a cationic surfactant (cetyltrimethylammonium bromide, CTAB) and a hydrotrope (sodium salicylate, NaSal). We obtained rigid cylindrical, spherical, and flexible cylindrical micelles at pH 7, 2, and 0, respectively, which agrees well with the experimental results of Umeasiegbu et al. (Langmuir 2016, 32, 655). By analyzing the different micellar structural properties, including distribution and molecular orientation of CTA+ and Sal- inside the micelle, we found that the binding form of the protonated salicylate molecules with CTA+ is different from that of Sal- ions. Because of the protonation of salicylate molecules with reduction in pH, their hydrogen bonding interactions with water molecules strengthened and the electrostatic interactions with CTA+ headgroups weakened. Thus, the repulsion of the CTA+ headgroups led to the breakage of the cylindrical micelle into spherical ones. At pH 0, the H-bond-strengthened cation-π interactions between salicylate and CTA+ were verified. We concluded that the penetration of salicylate molecules inside the micelle and the strong association of Cl- ions on the micellar surface play a key role in the formation of a flexible cylindrical micelle. This work provides an atomic-level insight into the mechanism of pH-induced shape transitions in the CTAB/NaSal systems, which is expected to be helpful to understand the aggregate behavior of cationic surfactant-hydrotrope solution.

A novel form of RNA double helix based on G·U and C·A+ wobble base pairing.

Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double-helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A+ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson–Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors.

Structural Study of Citrate Layers on Gold Nanoparticles: Role of Intermolecular Interactions in Stabilizing Nanoparticles

The structure of citrate adlayers on gold nanoparticles (AuNPs) was investigated. Infrared (IR) and X-ray photoelectron spectroscopy (XPS) analyses indicate citrate anions are adsorbed on AuNPs through central carboxylate groups. A unique structure of adsorbed citrate is determined, and a pH-induced structural transition is presented. IR analysis probes dangling dihydrogen anions (H2Citrate(-)) and hydrogen bonding of carboxylic acid groups between adsorbed and dangling citrate anions. A contribution of steric repulsion between citrate layers to particle stability is characterized. Structure-based modeling, which is consistent with scanning tunneling microscopy (STM) and transmission electron microscopy (TEM) images in the literature, suggests organization details relating to the formation of self-assembled layers on (111), (110), and (100) surfaces of AuNPs. Adsorption characteristics of the citrate layer include the interaction between hydrogen-bonded citrate chains, bilayer formation, surface coverage, and chirality. The enthalpic gain from intermolecular interactions and the importance of molecular structure/symmetry on the adsorption are discussed. Combining the enthalpic factor with surface diffusion and adsorption geometry of (1,2)-dicarboxyl fragments on Au(111), H2Citrate(-) anions effectively stabilize the (111) surface of the AuNPs. The detailed understanding of intermolecular interactions in the molecular adlayer provides insight for nanoparticle formation and stabilization. We expect these findings will be relevant for other nanoparticles stabilized by hydroxy carboxylate-based amino acids and have broad implications in NP-based interfacial studies and applications.

2P215 低いpHが誘起するDOPS/MO膜の一枚膜リポソームからキュービック相への構造転移の初期過程(13B. 生体膜・人工膜:ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

2P215 低いpHが誘起するDOPS/MO膜の一枚膜リポソームからキュービック相への構造転移の初期過程(13B. 生体膜・人工膜:ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

Effect of pH on turbidity, size, viscosity and the shape of sodium caseinate aggregates with light scattering and rheometry.

The characterization of sodium caseinate solutions as a function of pH was determined using titration with HCL through turbidimetry in different concentrations (0.03wt.%, 0.045wt.%, 0.06wt.%, 0.09wt.%, 0.2wt.%, and 0.3wt.%). Additionally, the coupling of slow in situ acidification of the solution and rheometry was utilized to gain deeper insights into pH-induced structural transitions during the self assembly process and particle size distribution analysis have been used to determine the behavior of sodium caseinate solutions in different pHs. The formation of aggregates during the acidification process was clearly visualized using microscopy. Surprisingly the viscosity of sodium caseinate solution at pH 4.64 was maximum and decreased by lowering pH. Particle size analysis confirmed the onset of big aggregates on decreasing pH but further acidification led to formation of smaller aggregates. A small concentration effect on pI was seen where at sodium caseinate levels of 0.03wt.% the pI occurred at 4.29, where at sodium caseinate levels of 0.30wt.% pI value was 4.64.

Self-assembly of β-lactoglobulin and the soluble fraction of gum tragacanth in aqueous medium

Spectrophotometric and light scattering measurements, along with optical microscopy, were used to follow the complexation and coacervation process that occur when β-lactoglobulin (BLG)/tragacanthin (T) mixed dispersions (0.3wt.% total concentration; BLG:T ratio of 2:1) were brought from pH 6 to pH 2. In addition, the coupling of slow in situ acidification of the mixture and rheometry was utilised to gain deeper insights into pH-induced structural transitions during the assembly process. The results obtained by this multi-methodological approach allowed the associative phase separation process to be parameterised in terms of a set of characteristic pH values (∼5.3, ∼4.8, ∼4.5, ∼4.15, ∼4, ∼3.8, ∼2.5) at which critical structural changes took place. Investigation of the absorbance profiles of complexed/coacervated systems as a function of time revealed that several transitions could occur at different time scales. Morphological changes in the assemblies and the subsequent formation of some flocculant substances during the late stage of process were clearly visualised using microscopy.

Biocompatible, pH-sensitive AB2 miktoarm polymer-based polymersomes: preparation, characterization, and acidic pH-activated nanostructural transformation

Motivated by the limitations of liposomal drug delivery systems, we designed a novel histidine-based AB(2)-miktoarm polymer (mPEG-b-(polyHis)(2)) equipped with a phospholipid-mimic structure, low cytotoxicity, and pH-sensitivity. Using "core-first" click chemistry and ring-opening polymerization, mPEG(2kDa)-b-(polyHis(29kDa))(2) was successfully synthesized with a narrow molecular weight distribution (1.14). In borate buffer (pH 9), the miktoarm polymer self-assembled to form a nano-sized polymersome with a hydrodynamic radius of 70.2 nm and a very narrow size polydispersity (0.05). At 4.2 µmol/mg polymer, mPEG(2kDa)-b-(polyHis(29kDa))(2) strongly buffered against acidification in the endolysosomal pH range and exhibited low cytotoxicity on a 5 d exposure. Below pH 7.4 the polymersome transitioned to cylindrical micelles, spherical micelles, and finally unimers as the pH was decreased. The pH-induced structural transition of mPEG(2kDa)-b-(polyHis(29kDa))(2) nanostructures may be caused by the increasing hydrophilic weight fraction of mPEG(2kDa)-b-(polyHis(29kDa))(2) and can help to disrupt the endosomal membrane through proton buffering and membrane fusion of mPEG(2kDa)-b-(polyHis(29kDa))(2). In addition, a hydrophilic model dye, 5(6)-carboxyfluorescein encapsulated into the aqueous lumen of the polymersome showed a slow, sustained release at pH 7.4 but greatly accelerated release below pH 6.8, indicating a desirable pH sensitivity of the system in the range of endosomal pH. Therefore, this polymersome that is based on a biocompatible histidine-based miktoarm polymer and undergoes acid-induced transformations could serve as a drug delivery vehicle for chemical and biological drugs.

X-ray Structure of Cerulean GFP: A Tryptophan-Based Chromophore Useful for Fluorescence Lifetime Imaging,

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.

PH-induced structural transitions of caseins

Caseins are relatively small (molecular mass ∼20 kDa), unstructured milk proteins of which the main components are referred to as α s-, β- and κ-casein. All three components lack a compact folded conformation, which can be ascribed to a combination of their low overall hydrophobicity and high net charge. Structural transitions of the three caseins in response to variation of pH were investigated using fluorescence and circular dichroism (CD) spectroscopy. Tryptophan emission parameters (intensity and wavelength of emission maximum) and CD spectra showed that at neutral and alkaline pH the caseins exist predominantly in random coil conformation. As the solvent is made acidic the added protons compensate the negative charges on the caseins and reduce the repulsion between like charged residues, allowing the casein chains to fold. At the pI (pH 4–5), the net charge on the protein tends to zero and the protein should approach its maximally structured state. Below pI, the uncompensated charges and their interactions reappear, resulting in slackening of the compact structure and formation of a partially unfolded intermediate. These conclusions were borne out by the biphasic pH-dependence of the fluorescence emission parameters of Trp as well as of ANS incubated with the caseins. Measurement of the efficiency of energy transfer between Trp (donor) and ANS (acceptor) and of the CD spectra of caseins as functions of pH were also consistent with this scenario.

Complex coacervation between β-lactoglobulin and Acacia gum: A nucleation and growth mechanism

Complex coacervation between proteins and polysaccharides is a demixing process mainly driven by electrostatic interactions. During this process many structural transitions occur, involving the formation of soluble complexes, aggregated complexes, and coacervates. The dynamic mechanism of complexation/coacervation was studied on β-lactoglobulin (BLG)/Acacia gum (AG) mixed dispersions (0.1 wt% total concentration; BLG:AG ratio of 2:1) using small angle static light scattering (SALS). Acidification of BLG/AG dispersions was induced by dissolution of 0.11 wt% glucono- δ-lactone, allowing in situ SALS measurements. Time evolution of turbidity, scattered light intensity at 46° scattering angle ( I 46 ) or slope of scattering functions at high q range revealed the existence of six pH-induced structural transitions. During BLG/AG complexation and before coacervation took place, scattering profiles displayed a monotonic decrease of I ( q ) as a function of q. A correlation peak in the scattering functions was only observed when coacervates appeared in the system. The wave vector q max corresponding to the maximum in scattered intensity first shifted toward larger q values, indicating an increasing number of coacervates, then shifted toward smaller q values, as a consequence of the system coarsening. The power laws q max ∼ t − α and I max ∼ t − β gave values of 1.9 and 9.2, respectively, values much larger than those expected for intermediate and late stages of spinodal decomposition. From these results, it was concluded that complex coacervation between BLG and AG was a nucleation and growth type process. In addition, the temporal evolution of I 46 followed power laws with two different exponents. First exponent corresponding to BLG/AG complexation was 3.0 ± 0.3 and indicated a diffusion-controlled growth mechanism. Second exponent corresponding to the initiation of phase separation to the coacervation process was 6.5 ± 0.3 and revealed an interfacially-controlled growth mechanism.

PH-Induced Structural Transitions during Complexation and Coacervation of β-Lactoglobulin and Acacia Gum

pH-Induced structural changes during complex coacervation between beta-lactoglobulin (BLG) and Acacia gum (AG) in aqueous solutions were determined by coupling slow in situ acidification of BLG/AG mixed dispersions and different experimental methods. The combined signal evolution of dynamic light scattering at 90 degrees scattering angle (I(90)), electrophoretic mobility, turbidimetry (tau), circular dichroism, and phase contrast microscopy allowed the distinction of critical structural transitions and the definition of their corresponding pH. The formation of soluble BLG/AG complexes was initiated at pH(sc) (4.90), since I(90) and tau significantly increased from the baseline. In parallel or just following complexation, a conformational change of BLG was detected at pH(pct) (4.8). An increase in positive charge density of BLG induced complex aggregation at pH(ca) (4.7). More efficient charge neutralization of aggregated complexes, especially through the lowering of the number of AG negative charges, promoted initiation of phase separation at pH(psi) (4.4). Mixed dispersions became unstable and phase separation occurred at pH(ps) (4.2). The phase separation of mixed dispersions was suggested by the maximum value of scattered light, by an important acceleration of the dispersion turbidity, by a strong increase of hydrodynamic radii, and by the first appearance of light fluctuations as observed by phase contrast microscopy. At the microscopic level, the first coacervates were observed at pH(coa) (4.0), near the pH of the maximum of turbidity. It was also noticed that, from the onset of interactions between biopolymers, the pH decrease led to (i) a gradual homogenization of particle size in the mixed dispersion as suggested by the decrease of dispersion polydispersity and (ii) conformational transitions of the protein (a loss of alpha-helix structure at pH(pct) and a gain in protein secondary structure near pH(coa), probably involving beta-sheet components).

Ionization Characteristics and Structural Transitions of Alternating Maleic Acid Copolymer Films

Alternating maleic acid copolymers dissolved in aqueous solutions exhibit characteristic two-step dissociation profiles and pH-induced structural transitions in dependence on the comonomer unit. In extension of these findings, we analyzed a set of thin films of maleic acid copolymers (poly(octadecene-alt-maleic acid), POMA; poly(propylene-alt-maleic acid), PPMA; poly(styrene-alt-maleic acid), PSMA) randomly attached to planar glass surfaces by covalent binding. Streaming potential/streaming current measurements with a microslit electrokinetic setup (MES) were applied to determine zeta potential (ζ) and surface conductivity (Kσ) data of the polymer layers in aqueous solutions of varied pH. The results confirmed the two-step dissociation behavior for all immobilized copolymers. A dramatic increase of Kσ was observed for PPMA and PSMAbut not for POMAlayers at alkaline pH values, indicating that electrostatic repulsion of ionized groups controls the extension of the confined layers as long as hydrophobic inte...

The role of acidic amino acid residues in the structural stability of snake cardiotoxins.

We have recently shown that membrane-related activities of cardiotoxin V from Naja naja atra (CTX A5) are diminished at acidic pH although the overall beta-sheet structure of the molecule is maintained. In order to understand more about the mechanism of inactivation of CTX at acidic pH, we studied the effect of pH and denaturing reagents on the structural stability of CTX. We found, first, pH-induced structural transitions occurred in CTX A5 at two pH values as judged by the CD ellipticity around 195 nm: an increase in the beta-sheet content occurred around pH 4 and followed by a decrease, therein, around pH 2. The pKa of three acidic amino acid residues in CTX A5, i.e., Glu-17, Asp-42, and Asp-59, were determined to be 4.0, 3.2, and below 2.3, respectively, by NMR spectroscopy. The low pKa value of Asp-59 implies salt bridge formation between Lys-2 and Asp-59. Thus, electrostatic interaction may stabilize the three loop structure in addition to the hydrogen bonds between N- and C-termini of CTX molecule. Second, 2,2,2-trifluoroethanol (TFE) and guanidinium chloride (GdmHCI) were found to induce alpha-helical and random coil formation, respectively, in CTX A5 and eight other beta-sheet CTXs. Comparison of the relative potencies of TFE and GdmHCI to induce structural changes suggests that the amino acid residue located at position 17 plays a role in the structural stability. Specifically, CTXs containing negatively charged Glu-17 are least stable. It is suggested that Glu-17 may perturb the interaction between Lys-2 and Asp-59, and thus the overall stability of beta-sheet, in the presence of denaturing reagent. In conclusion, the perturbed structural stability of CTXs may partially explain the lower activity CTX exhibits at acidic pH. A structural model to account for the unfolding and refolding of CTX molecules without the breaking of disulfide bonds is also proposed.

Versatility of heme coordination demonstrated in a fungal peroxidase. Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245-->Asn mutant at various pH values.

The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly characteristic of a 6-coordinate high-spin heme, presumably with a water molecule bound on the distal side of the Fe atom. The pKa of the alkaline transition of the mutant is much lower than that of the wild-type protein. At pH 10.0 the D245N mutant is in its final alkaline form, which markedly differs from that of the parent enzyme. The spectral data indicate that the majority of the protein has 5-coordinate high-spin heme (5-c HS), with the Fe-His 183 bond broken and the distal axial coordination site of the heme iron occupied by a hydroxyl group, which is strongly hydrogen-bonded with distal Arg51. Therefore, the Asp245-->Asn mutation on the proximal side results in the breakage of the Fe-His bond at alkaline pH.