The role of histidine-42 in the oxidation-reduction mechanism of Chromatium vinosum high potential iron-sulfur protein

The second order rate constants for the oxidation of high potential iron-sulfur protein (Hipip) of Chromatium vinosum by ferricyanide were determined as a function of ionic strength and pH. From the ionic strength results, calculations were done to correct the rate constant at each pH for the electrostatic interactions between Hipip and ferricyanide. The electrostatic corrections are necessary since the charge of the protein changes as a function of pH and can mask the ionization of mechanistically important amino acid residues. An apparent pK_a 7 was obtained from electrostatically corrected rate-pH profile, indicating the possible participation of histidine. Perturbation difference spectroscopic studies of Hipip as a function of pH also gave apparent pK_a values of 6.9 and 6.7 for the reduced and oxidized protein, respectively. That it was indeed His 42 (the only His in the polypeptide) that was responsible for the kinetic and spectroscopic pK_a values was demonstrated by modification of His 42 of Hipip by the histidine selective reagent diethylpyrocarbonate. No modification of Tyr 19 could be detected. It is concluded that either deprotonation or modification of His 42 results in the destabilization of the reduced cluster and thus a faster rate of oxidation. This work provides the first experimental evidence of the ‘squeeze effect’ mechanism (Carter, C.W., Jr., Kraut, J., Freer, S.T. and Alden, R.A. (1974) J. Biol. Chem. 249, 6339–6346) in which the polypeptide directly modulates the stability of the iron-sulfur cluster.     

Acid dissociation constants of diphytanylglycerolphosphorylglycerol-methylphosphate, and diphytanylglycerolphosphorylglycerophosphate and its deoxy analog

Acid dissociation constants of 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-methylphosphate (PGP-Me), the major phospholipid in extreme halophiles (Halobacteriaceae), and of the demethylated 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-phosphate (PGP) and its deoxy analog 2,3-diphytanyl-sn-glycero-1-phosphoryl-1′-(1′,3′-propanediol-3′-phosphate) (dPGP), were calculated by an original mathematical procedure from potentiometric titration data in methanol/water (1:1, v/v) and found to be as follows: for PGP-Me (and presumably PGP), pK₁=3.00 and pK₂=3.61; for PGP, pK₃=11.12; and for dPGP, pK₁=2.72, pK₂=4.09, and pK₃=8.43. High-resolution ³¹P NMR spectra of intact PGP-Me in benzene/methanol or in aqueous dispersion showed two resonances corresponding to the two P-OH groups, each of which exhibited a chemical shift change in the pH range 2.0–4.5, corresponding to acid dissociation constants pK₁=pK₂=3.2; no further ionization (pK₃) was detected at higher pH values in the range 5–12. The present results show that PGP-Me titrates as a dibasic acid in the pH range 2–8, but above pH 8, it titrates as a tribasic acid, presumably PGP, formed by hydrolysis of the methyl group during the titration with KOH. Calculation of the concentrations of the ionic molecular species of PGP-Me, PGP and dPGP as a function of pH showed that the dianionic species predominate in the pH range 5–9, covering the optimal pH for growth of Halobacteriaceae. The results are consistent with the concept that the hydroxyl group of the central glycerol moiety in PGP-Me and PGP is involved in the formation of an intramolecular hydrogen-bonded cyclic structure of the polar headgroup, which imparts greater stability to the dianionic form of PGP-Me and PGP in the pH range 5–9 and facilitates lateral proton conduction by a process of diffusion along the membrane surface of halobacterial cells.     

Intermolecular Re---H·H---X hydrogen bonding (X = N, C) involving ReH5(PPh3)3

Ab initio (B3LYP) calculations show that PD·H---ReH₄(PH₃)₃ (PD = Proton donor) interactions follow the order PD = pyrrole > NH₃ > HCCH > C₂H₄ > CH₃---H 0 and decrease with the pK_a of the PD. For equivalent pK_a's, NH interacts more strongly than CH. However, intermolecular hydrogen-bonding of the M---H·H---C type is too weak to be detected experimentally in FTIR or UV-vis studies between ReH₅(PPh₃)₃ and PhCCH, C₆F₅H or PhCHCl₂.     

The effects of temperature and pH on the reaction between 1-chloro-2,4-dinitrobenzene and glutathione, catalyzed by the major glutathione S-transferase from Galleria mellonella

The nucleophilic substitution reaction between glutathione and 1-chloro-2,4-dinitrobenzene has been studied at temperatures between 4 and 42°C and pH values between 6.99 and 10.80. The apparent enthalpy, entropy and free energy of ionization of the thiol group have been estimated as have the apparent enthalpy, entropy and free energy of activation of the reaction between the glutathione thiolate anion and the aromatic electrophile. The results obtained permit the calculation of values of the second order rate constant governing the reaction at a range of temperatures and pHs. These values are in accord with those reported in the literature from experimental work by others. The major glutathione S-transferase from Galleria mellonella has been studied with respect to its kinetic responses to changes in pH and temperature. There appear to be two kinetically critical ionizations governing the reaction at high pH. These ionization events are characterized by apparent pK_a values of 8.61 ± 0.15 and 9.16 ± 0.22. A thermodynamic model of the kinetic behavior of the enzyme permits the prediction of its activity over a range of pH and temperature values. The apparent free energy of activation for the enzyme catalyzed reaction is only 7% lower than that for the non-catalyzed reaction between 1-chloro-2,4-dinitrobenzene and glutathione thiolate anion. This observation is compatible with the suggestion that promotion of the ionization of the glutathione thiol group is the major mechanism of catalysis.     

Synthesis and characterization of a dianionic carbohydrate-based phospholipid

A novel carbohydrate-based phospholipid containing a phosphatidic acid head group, bis-(2,3-lauroyl)-1-methoxy-5-ribo-phosphatidic acid (DLRPA), was synthesized and characterized by ¹H NMR, ¹³C NMR, and ³¹P NMR. This molecule is an analog of dilauroyl phosphatidic acid (DLPA). The T_m of DLRPA decreases with increasing pH in a similar pattern to DLPA, as determined by MDSC. From this thermotropic behavior, the apparent pK_as of DLRPA are estimated to be 4 and 8.     

Relationship between pKa of 8-quinolinol derivatives and a π-donor ability of the 8-quinolinolato oxygen in linear nitrosylruthenium(II) complexes

The relationship between the pK_a of 8-quinolinol derivatives {8-quinolinol (Hqn), 2-methyl- (H2-Meqn), 2,4-dimethyl- (H2,4-diMeqn), 5-chloro- (H5-Clqn) and 5,7-dichloro-8-quinolinols (H5,7-diClqn)} and a π-donor ability of the 8-quinolinolato oxygens has been investigated by the identification of the structures of the major products, [RuCl(QN)(QN′)NO] (HQN=8-quinolinol derivative; HQN′=different 8-quinolinol derivatives), obtained by the reaction of [RuCl₃(QN or QN′)NO]⁻ with HQN′ or HQN. The results obtained clearly showed that the oxygen of the 8-quinolinol derivative that has a higher pK_a predominantly coordinates in the trans position to the NO ligand and is a better π-electron donor. The order of the π-electron donor ability for the oxygen of the 8-quinolinol derivatives is as follows: H2-Meqn≥H2,4-diMeqn>Hqn≥H5-Clqn>H5,7-diClqn, almost agreeing with the magnitude of the pK_a values of the corresponding 8-quinolinols. The structures of cis-1 [RuCl(5,7-diClqn)₂NO] and cis-1 [RuCl(5,7-diClqn)(2-Meqn)NO] were determined by X-ray diffraction.     

Study of the swelling dynamics with overshooting effect of hydrogels based on sodium alginate-g-acrylic acid

A series of hydrogels were synthesized by graft cross-link copolymerization of sodium alginate (SA) and acrylic acid (AA) using N, N-methylene-bis-(acrylamide) as a cross-linker. By study of the swelling kinetics of the hydrogels in different buffer solutions, the overshooting effect was observed in acidic medium, namely the gels firstly swelled to a maximum value following by a gradual deswelling until the equilibrium. The phenomenon is interpreted as a cooperative physical cross-linking caused by the hydrogen bond formation between the carboxyl groups of the hydrogels in a hydrophobic environment. The hydrogen bond formation was further confirmed by FT-IR spectra. The dependence of overshooting effect on the pH of buffer solution was more noticeable in comparison with the composition of hydrogels, demonstrating that the cooperative physical cross-linking caused by the hydrogen bond formation is dominant. Whether or not the overshooting effect appears is not only relative to the pH of buffer solution, but also depends on the pK_a of carboxyl groups on the network. The overshoot processes of the hydrogels under acidic medium at pH below the pK_a follow a quantitative model proposed by Díez-Peńa et al., and the theoretical curves are in very good agreement with the experimental data. While in pH > pK_a buffer solutions, the overshoot phenomenon does not appear arising from the repulsive interaction between the ionized carboxyl groups, the swelling processes follow Schott second-order rate equation.     

Comparative molecular field analysis and QSAR on substrates binding to cytochrome p450 2D6

In this study, we utilized comparative molecular field analysis (CoMFA) to gain a better understanding of the steric and electrostatic features of the cytochrome P450 2D6 (CYP2D6) active site. The training set consists of 24 substrates with reported K_M values from liver microsomal CYP2D6 spanning an activity range of almost three log units. The low energy conformers were fit by root mean square (RMS) to minaprine at the site of metabolism and to the protonated nitrogen. In this manner, we constructed two CoMFA models, one model with a distance constraint and another without. The model with the distance parameter (non-crossvalidated R²=0.99) was approximately equal to the CoMFA without a distance parameter (non-cross-validated R²=0.98). Validation of our CoMFA was accomplished by predicting the K_M values of 15 diverse CYP2D6 substrates not in the original training set resulting in a predictive R²=0.62. Finally, we also pursued correlations of pK_a and log P with CYP2D6 substrate K_M in an effort to investigate other physicochemical properties.     

The One-Electron Reduction Potential of 3-Amino-1, 2, 4-benzotriazine 1, 4-dioxide (Tirapazamine): A Hypoxia-Selective Bioreductive Drug

The one-electron reduction potential of 3-amino-l, 2, 4-benzotriazine 1, 4-dioxide, tirapazamine (SR 4233) in aqueous solution has been determined by pulse radiol-ysis. Reversible electron transfer was achieved between radiolytically-generated one-electron reduced radicals of tirapazamine (T), and quinones or benzyl viologen as redox standards. The reduction potential E_m7(T/T^±) was -0.45 ± 0.01 V vs. NHE at pH 7. From the pH dependence of the reduction potential, pK_a = 5.6 ± 0.2 was estimated for the tirapazamine radical, a value similar to the pK_a determined by other methods.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid–base buffers

We have previously demonstrated that a solid-state buffer could be successfully used to control the ionization state of subtilisin Carlsberg cross-linked microcrystals (CLECs) suspended in supercritical ethane (sc-ethane) in the presence of acid–base active species such as salt hydrates and zeolite molecular sieves. Here we studied the effect of six zwitterionic proton/sodium (pH–pNa) solid-state acid–base buffers on the catalytic activity of subtilisin CLECs in sc-ethane at high and low water activity (a_W). CLECs were strongly activated by increasing a_W. At high a_W, and despite the high hydrolysis rates, transesterification activities were still about one order of magnitude higher than those observed at lower a_W. This is in contradiction with what was previously reported in the absence of acid–base control and supports the hypothesis that the poor catalytic performance of subtilisin CLECs at high a_W observed in those studies was due to the inhibitory effect of the hydrolytic by-product, rather than to the competition of water with propanol for the acyl-enzyme intermediate. Although the catalytic activity of subtilisin showed a general positive correlation with the aqueous pK_a of the acid–base buffers tested here, our results also show that as expected, the acid–base behavior of the buffers in nonaqueous media is more complex than what can be predicted from aqueous-based parameters alone. This work further confirms the usefulness of solid-state acid–base buffers in supercritical biocatalysis but highlights the need for further research on the topic.     

The pH dependence of flavivirus envelope protein structure: insights from molecular dynamics simulations

The flavivirus membrane fusion is triggered by the acid pH of the endosomes after virus endocytosis. The proposed mechanism involves changes in the protonation state of conserved histidine residues of the E protein present in the viral surface that undergoes a series of structural rearrangements that result in the fusion between the endosome and viral bilayers. We studied the pH dependence of E protein rearrangements of dengue virus type 2, used as a model, in the pH range experimented by the virus along the fusion process. We employed a low computational cost scheme to explore the behavior of the E protein by molecular dynamics (MD) simulations of complete systems that include the protein, the solvent, and ions. The procedure alternates cyclically the update of the ionization states of the protein residues with common MD steps applied to the new ionization configuration. Important pH-dependent protein structure rearrangements consistent with the changes of the protonation states of conserved histidine residues were observed. The involvement of other conserved residues in the flavivirus in the rearrangements was also identified. The results show interesting correlations with a proposed model for the fusion mechanism, as well as the experimentally identified key residues, contributing to a better understanding of the structural changes in protein E that lead to the fusion process.     

Nuclear magnetic resonance titration curves of histidine ring protons. Conformational transition affecting three of the histidine residues of ribonuclease.

NMR titration curves are reported for the 4 histidine residues of ribonuclease A in sodium acetate and for ribonuclease S in sodium acetate, phosphate, and sulfate solutions. Evidence is presented that the imidazole side chain of histidine residue 48 undergoes a conformational change, probably also involving the carboxyl side chain of aspartic acid residue 14. This group is considered to be responsible for the low pH inflection with pKa 4.2 present in the NMR titration curve of the C-2 proton resonance of histidine 48. The NMR titration curves of the active site histidine residues 12 and 119 also exhibit inflections at low pH values, although there is no carboxyl group within 9 A of the imidazole side chain of histidine residue 12 in the structure of ribonuclease S determined by x-ray crystallography (Wyckoff, H. W., Tsernoglou, D., Hanson, A. W. Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305-328). Curve fitting was carried out on 11 sets of NMR titration data using a model in which the 3 histidine residues 12, 119, and 48 are assumed to be affected by a common carboxyl group. The results obtained indicate that such a model with fewer parameters gives as good a representation of the data as the model in which each histidine residue is assumed to interact separately with a different carboxyl group. Therefore, it is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48. A model assuming mutual interaction of the active site histidine residues does not account for the low pH inflections in these curves.     

Preparation of tetrahydroimidazo[2,1-a]isoquinolines and their use as inhibitors of gastric acid secretion

A series of novel tetrahydroimidazo[2,1-a]isoquinolines was prepared based on a hetero Diels–Alder reaction between an enamine and 1,2,4-triazine as key step. A structure–activity relationship was established focussing on the influence of the substitution pattern in position 3 and 6 of the heterocycle on antisecretory activity, lipophilicity, and pK_a value. Potent inhibitors of the gastric acid pump were identified.     

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate.

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.     

Enzymatic properties of a novel highly active and chelator resistant protease from a Pseudomonas aeruginosa PD100

Pseudomonas aeruginosa PD100 capable of producing an extracellular protease was isolated from the soil collected from local area (garbage site) from Shivage market in Pune, India. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 36 kDa on SDS-PAGE. The optimum pH value and temperature range were found to be 8 and 55–60 °C, respectively. The enzyme exhibited broad range of substrate specificity with higher activity for collagen. The enzyme was inhibited with low concentration of Ag²⁺, Ni²⁺, and Cu²⁺. β-Mercaptoethanol was able to inactivate the enzyme at 2.5 mM, suggesting that disulfide bond(s) play a critical role in the enzyme activity. Studies with inhibitors showed that different classes of protease inhibitors, known to inhibit specific proteases, could not inhibit the activity of this protease. Amino acid modification studies data and pK_a values showed that Cys, His and Trp were involved in the protease activity. P. aeruginosa PD100 produces one form of protease with some different properties as compared to other reported proteases from P. aeruginosa strains. With respect to properties of the purified protease such as pH optimum, temperature stability with capability to degrade different proteins, high stability in the presences of detergents and chemicals, and metal ions independency, suggesting that it has great potential for different applications.     

Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine

The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D(parallel)/D(perpendicular) of 1.31 and an effective correlation time (4D(perpendicular) + 2D(parallel))(-1) of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 degrees C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.     

Acid-base and metal ion-binding properties of flavin mononucleotide (FMN). Is a ‘dielectric’ effect responsible for the increased complex stability?

Due to contradictions in the literature we have redetermined the acid-base properties of riboflavin (=RiFl; vitamin B₂), i.e. 7,8-dimethyl-10-ribityl-isoalloxazine, and of flavin mononucleotide (FMN²⁻), also known as riboflavin 5′-phosphate, via potentiometric pH titrations (I = 0.1 M, NaNO₃; 25 °C). In contrast to various claims, the isoalloxazine ring cannot be protonated at pH > 1, a result in agreement with an early study (pK_a = −0.2; L. Michaelis, M.P. Schubert and C.V. Smythe, J. Biol. Chem., 116 (1936) 587–607); deprotonation of the ring system occurs in both compounds with pK_a 10. The pK_a value of 0.7 determined for the deprotonation of H₂(FMN) must be attributed to the release of the first proton from the fully protonated phosphate group; its second proton is released with pK_a = 6.18 in agreement with the acidity constants of various other monoprotonated monophosphate esters. The stability constants of the 1:1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Cd²⁺ (---M²⁺) and FMN²⁻ were determined by potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO₃; 25 °C). The log stability constants of all these M(FMN) complexes are about 0.2 log units higher than expected from the basicity of the phosphate group. This slight stability increase cannot be attributed to the formation of a seven-membered chelate involving the ribit-hydroxy group at C-4′ as the stability constants for the M²⁺ 1:1 complexes of glycerol 1-phosphate (G1P²⁻) demonstrate: G1P²⁻ contains the same structural unit which would also allow in this case the formation of the mentioned seven-membered chelate; however, the stability of the M(G1P) complexes is solely determined by the basicity of the phosphate group. Hence, in agreement with earlier conclusions (J. Bidwell, J. Thomas and J. Stuehr, J. Am. Chem. Soc., 108 (1986) 820–825) regarding Ni(FMN) one must conclude that the slight stability increase of the M(FMN) complexes has to be attributed to the isoalloxazine ring. The equality of the stability increase of the complexes for all the mentioned ten metal ions precludes its attribution to an interaction with an N site and makes a specific interaction with an O site also somewhat unlikely. In addition, carbonyl oxygens appear as not very favorable for the formation of macrochelates by a further interaction with already phosphate-coordinated metal ions. Therefore, we propose that the slight but significant stability increase originates from M(FMN) species (with a formation degree of about 30%) in which the hydrophobic flavin residue is close to the metal ion, thereby lowering the ‘effective’ dielectric constant in the microenvironment of the metal ion and thus indirectly promoting the −PO₃²⁻/M²⁺ interaction.     

不同动力学分馏系数对北京山区侧柏叶片水δ18O的模拟

利用稳定同位素技术对植物叶片水¹⁸O同位素组成(δ_L,b)进行研究,可以为植物叶片生理及森林水文的研究提供理论参考。本研究连续监测北京山区侧柏人工林生态系统冠层大气水汽浓度(W_a)和大气水汽¹⁸O同位素值组成(δ_v),结合测定的侧柏枝条水¹⁸O同位素组成(δ_x)和δ_L,b,分析了动力学分馏系数ε_k1(32%)和ε_k2(28%)对δ_L,b的预测效果。结果表明: 侧柏人工林生态系统W_a日变化无明显规律,大气相对湿度(RH)日变化呈“V”型,气孔导度(g_s)在日尺度上先增大后减小;同位素接近稳态时(正午前后),δ_L,b略有增加,W_a、RH、g_s与δ_L,b均呈显著负相关关系;同位素接近稳态条件下,将不同动力学分馏系数ε_k1、ε_k2应用于Craig-Gordon模型,预测δ_L,b,ε_k2的预测值更接近δ_L,b的实测值,表明ε_k2应用于模型更符合北京山区侧柏叶片水同位素富集情况。研究结果将加深对叶片水同位素富集模型、蒸散拆分模型的认识。     

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g_XXX = 6.0 and ^g= 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g_XXX = 2.38 and _g = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.