Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.     

鼠脑红蛋白突变体F106L与氰根的结合作用

构建了鼠脑红蛋白(Mouse neuroglobin)的突变体F106L, 以探求近端残基对脑红蛋白血红素口袋结构的贡献. 通过溶液核磁共振方法研究了外来配体氰根离子与NgbF106L蛋白的结合作用, 结果显示, 此结合存在动力学过程, 并且NgbF106LCN 突变蛋白氰根络合物可以可逆地释放氰根离子, 并使原来的第6配体His64(E7)又结合回到血红素铁上. 研究结果揭示, G5(Phe106)残基对脑红蛋白血红素构象而言较为保守; QM/MM结构优化结果表明, 位于G5 和FG5的近端残基对蛋白结构稳定性具有重要作用, 并可调控外来配体与蛋白作用的配位平衡与热动力学性质.     

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam). Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.     

How do azoles inhibit cytochrome P450 enzymes? A density functional study

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine. These structural motifs represent typical azoles, such as econazole, fluconazole, and metyrapone. The calculations show that azole binding is a stepwise mechanism whereby first the water molecule from the resting state of P450 is released from the sixth binding site of the heme to create a pentacoordinated active site followed by coordination of the azole nitrogen to the heme iron. This process leads to the breaking of a hydrogen bond between the resting state water molecule and the approaching inhibitor molecule. Although, formally, the water molecule is released in the first step of the reaction mechanism and a pentacoordinated heme is created, this does not lead to an observed spin state crossing. Thus, we show that release of a water molecule from the resting state of P450 enzymes to create a pentacoordinated heme will lead to a doublet to quartet spin state crossing at an Fe-OH(2) distance of approximately 3.0 A, while the azole substitution process takes place at shorter distances. Azoles bind heme with significantly stronger binding energies than a water molecule, so that these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding and the catalysis of substrate oxidation. Perturbations within the active site (e.g., a polarized environment) have little effect on the relative energies of azole binding. Studies with an extra hydrogen-bonded ethanol molecule in the model, mimicking the active site of the CYP121 P450, show that the resting state and azole binding structures are close in energy, which may lead to chemical equilibrium between the two structures, as indeed observed with recent protein structural studies that have demonstrated two distinct azole binding mechanisms to P450 heme.     

Resolution of chiral phosphate, phosphonate, and phosphinate esters by an enantioselective enzyme library

An array of 16 enantiomeric pairs of chiral phosphate, phosphonate, and phosphinate esters was used to establish the breadth of the stereoselective discrimination inherent within the bacterial phosphotriesterase and 15 mutant enzymes. For each substrate, the leaving group was 4-hydroxyacetophenone while the other two groups attached to the phosphorus core consisted of an asymmetric mixture of methyl, methoxy, ethyl, ethoxy, isopropoxy, phenyl, phenoxy, cyclohexyl, and cyclohexoxy substituents. For the wild-type enzyme, the relative rates of hydrolysis for the two enantiomers ranged from 3 to 5.4 x 10(5). Various combinations of site-specific mutations within the active site were used to create modified enzymes with alterations in their enantioselective properties. For the single-site mutant enzyme, G60A, the stereoselectivity is enhanced relative to that of the wild-type enzyme by 1-3 orders of magnitude. Additional mutants were obtained where the stereoselectivity is inverted relative to the wild-type enzyme for 13 of the 16 pairs of enantiomers tested for this investigation. The most dramatic example was obtained for the hydrolysis of 4-acetylphenyl methyl phenyl phosphate. The G60A mutant preferentially hydrolyzes the SP-enantiomer by a factor of 3.7 x 10(5). The I106G/F132G/H257Y mutant preferentially hydrolyzes the RP-enantiomer by a factor of 9.7 x 10(2). This represents an enantioselective discrimination of 3.6 x 10(8) between these two mutants, with a total of only four amino acid changes. The rate differential between the two enantiomers for any given mutant enzyme is postulated to be governed by the degree of nonproductive binding within the enzyme active site and stabilization of the transition state. This hypothesis is supported by computational docking of the high-energy, pentavalent form of the substrates to modeled structures of the mutant enzyme; the energies of the docked transition-state analogues qualitatively capture the enantiomeric preferences of the various mutants for the different substrates. These results demonstrate that the catalytic properties of the wild-type phosphotriesterase can be exploited for the kinetic resolution of a wide range of phosphate, phosphonate, and phosphinate esters and that the active site of this enzyme is remarkably amenable to structural perturbations via amino acid substitution.     

Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states

Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R(2) relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by (15)N nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme-substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.     

Roles of the proximal heme thiolate ligand in cytochrome p450(cam).

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450(cam) (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixture of high and low spin species. Low temperature (10 degrees C), low enzyme concentration (1 microM), high camphor concentration (1 mM), and 5--50 mM buffer concentrations increase the high to low spin ratio, but under no conditions examined was the protein more than 60% high spin. The C357H mutant has a poorer K(m) for camphor (23 vs 2 microM) and a poorer K(d) for putidaredoxin (50 vs 20 microM) than wild-type P450(cam). The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greater peroxidase activity. Electron transfer from putidaredoxin to the mutant is much slower than to the wild-type even though redox potential measurements show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O--O bond cleavage by P450 enzymes and also demonstrate that this ligand satisfies important roles in protein folding, substrate binding, and electron transfer.     

Thermal and Light-Induced Spin Transition in the High- and Low-Temperature Structure of [Fe(0.35)Ni(0.65)(mtz)(6)](ClO(4))(2)

The thermal and light induced spin transition in [Fe(0.35)Ni(0.65)(mtz)(6)](ClO(4))(2) (mtz = 1-methyl-1H-tetrazole) was studied by (57)Fe M?ssbauer spectroscopy and magnetic susceptibility measurements. In addition to the spin transition of the iron(II) complexes the compound undergoes a structural phase transition. The high-temperature structure could be determined by X-ray crystallography of the isomorphous [Fe(0.25)Ni(0.75)(mtz)(6)](ClO(4))(2) complex at room temperature. The X-ray structural analysis shows this complex to be rhombohedric, space group R&thremacr;, with a = 10.865(2) ? and c = 23.65(1) ? with three molecules in the unit cell. The transition to the low-temperature structure occurs at approximately 60 K without changing the spin state of the molecules. By subsequent heating of the complex the high-temperature structure is reached again between ca. 170 and 200 K. The spin transition behavior is strongly influenced by the structural changes, and the observed spin transition curves are completely different for the high- and low-temperature phases. In the high-temperature structure a complete and gradual spin transition between 220 and 120 K (T(1/2)(gamma(HS) = 0.5) = 185 K) is detected; the high-spin (HS) state is represented by one HS doublet in the M?ssbauer spectra. In the low-temperature structure a two-step transition curve is detected in the heating mode. About 36% of the molecules show a LS (low-spin) --> HS transition between ca 50 and 75 K. Then the HS fraction stays constant up to 150 K. A further increase in the high-spin fraction is observed at temperatures above 150 K. In this structural phase the HS state is represented by two different HS doublets in the M?ssbauer spectra. The formation of metastable HS states by making use of the LIESST effect is only possible in the low-temperature structure. By excitation of the LS molecules with green light, two different HS states are populated which show very different relaxation behavior. One HS state shows a relaxation to the LS state even at 10 K; the other HS state shows a very slow HS --> LS relaxation at 60 K (within days), leading to the HS fraction corresponding to the thermal equilibrium value.     

Electronic ground states of iron porphyrin and of the first species in the catalytic reaction cycle of cytochrome P450s

Electronic structures of iron(II) and iron(III) porphyrins are studied with density functional theory (DFT) using the GGA exchange functional OPTX in combination with the correlation functional PBE (OPBE) and with the correlation functional Perdew (OPerdew) together with a triple zeta-type basis set. These functionals, known for accurately predicting the spin ground state of iron complexes, are evaluated against other functionals for their performance in calculating relative energies for the various electronic states of both the iron porphyrins. The calculated energy orderings are triplet < quintet < singlet for the iron(II) porphyrin and quartet < sextet < doublet for the iron(III) porphyrin cation. Complexation by a thiolate ion (SH-) changes the preferred ground state for both species to high spin. This thiolate complex is used as a mimic for the cytochrome P450s active site to model the first step of the catalytic cycle of this enzyme. This first step is believed to concern the removal of an axial oxygen donating ligand from the hexacoordinated aqua-thiolate-porphyrin-iron(III) resting state. The DFT results suggest that this is not a free water molecule, because of its repulsive nature, but that it has instead hydroxy anion character. These calculations are in line with the experimentally observed change in the spin state from low to high spin upon this removal of the axial hydroxo ligand by binding of the substrate in the heme pocket of cytochrome P450.     

Exciplex Formation in Lipid-bound Escherichia coli Flavohemoglobin

Flavohemoglobins have the particular capability of binding unsaturated and cyclopropanated fatty acids as free acids or phospholipids. Fatty acid binding to the ferric heme results in a weak but direct bonding interaction. Ferrous and ferric protein, in presence or absence of a bound lipid molecule, have been characterized by transient absorption spectroscopy. Measurements have been also carried out both on the ferrous deoxygenated and on the CO bound protein to investigate possible long-range interaction between the lipid acyl chain moiety and the ferrous heme. After excitation of the deoxygenated derivatives the relaxation process reveals a slow dynamics (350 ps) in lipid-bound protein but is not observed in the lipid-free protein. The latter feature and the presence of an extra contribution in the absorption spectrum, indicates that the interaction of iron heme with the acyl chain moiety occurs only in the excited electronic state and not in the ground electronic state. Data analysis highlights the formation of a charge-transfer complex in which the iron ion of the lipid-bound protein in the expanded electronic excited state, possibly represented by a high spin Fe III intermediate, is able to bind to the sixth coordination ligand placed at a distance of at 3.5 Å from the iron. A very small nanosecond geminate rebinding is observed for CO adduct in lipid-free but not in the lipid-bound protein. The presence of the lipid thus appears to inhibit the mobility of CO in the heme pocket.     

Hydrogen‐Bonding Interactions Trigger a Spin‐Flip in Iron(III) Porphyrin Complexes

A key step in cytochrome P450 catalysis includes the spin‐state crossing from low spin to high spin upon substrate binding and subsequent reduction of the heme. Clearly, a weak perturbation in P450 enzymes triggers a spin‐state crossing. However, the origin of the process whereby enzymes reorganize their active site through external perturbations, such as hydrogen bonding, is still poorly understood. We have thus studied the impact of hydrogen‐bonding interactions on the electronic structure of a five‐coordinate iron(III) octaethyltetraarylporphyrin chloride. The spin state of the metal was found to switch reversibly between high (S=⁵/₂) and intermediate spin (S=³/₂) with hydrogen bonding. Our study highlights the possible effects and importance of hydrogen‐bonding interactions in heme proteins. This is the first example of a synthetic iron(III) complex that can reversibly change its spin state between a high and an intermediate state through weak external perturbations.     

Light- and thermal-induced spin crossover in [Fe(abpt)2(N(CN)2)2]. Synthesis, structure, magnetic properties, and high-spin<-->low spin relaxation studies

[Fe(abpt)2(N(CN)2)2] (abpt = 4-amino-3,5-bis(pyridin-2-yl)-1,2,4-triazole) represents the first example of an iron(II) spin-crossover compound containing dicyanamide ligand, [N(CN)(2)](-), as a counterion. It shows an incomplete two-step spin transition with around 37% of HS molecules trapped in the low-temperature region when standard cooling or warming modes, i.e., 1-2 K min(-)(1), were used. The temperature, T(1/2) approximately 86 K, at which 50% of the conversion takes place, is one of the lowest temperatures observed for an iron(II) spin-crossover compound. Quenching experiments at low temperatures have shown that the incomplete character of the conversion is a consequence of slow kinetics. The quenched HS state relaxes back to the LS state displaying noticeable deviation from a single-exponential law. The rate of relaxation was evaluated in the range of temperatures 10-60 K. In the upper limit of temperatures, where thermal activation predominates, the activation energy and the pre-exponential parameter were estimated as E(a) approximately 280 cm(-)(1) and A(HL) approximately 10 s(-)(1), respectively. The lowest value of k(HL) around 1.2 x 10(-)(4) s(-)(1) (T = 10 K) was obtained in the region of temperatures where tunneling predominates. A quantitative light induced excited spin state trapping (LIESST) effect was observed, and the HS --> LS relaxation in the range of temperatures 5-52.5 K was studied. From the Arrhenius plot the two above-mentioned characteristic regimes, thermal-activated (E(a) approximately 431 cm(-)(1) and A(HL) approximately 144 s(-)(1)) and tunneling (k(HL) approximately 1.7 x 10(-)(6) s(-)(1) at 5 K), were characterized. The crystal structure was solved at room temperature. It crystallizes in the triclinic P_1 space group, and the unit cell contains a centrosymmetric mononuclear unit. Each iron atom is in a distorted octahedral environment with bond distances Fe-N(1) = 2.216(2) A, Fe-N(2) = 2.121(2) A, and Fe-N(3) = 2.160(2) A for the pyridine, triazole, and dicyanamide ligands, respectively.     

Chemical rescue of the distal histidine mutants of tryptophan 2,3-dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.     

Posttranslational hydroxylation of human phenylalanine hydroxylase is a novel example of enzyme self-repair within the second coordination sphere of catalytic iron

Phenylalanine hydroxylase, a mononuclear non-heme iron enzyme, catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and reduced pterin cofactor. X-ray structural studies have established the coordination around the iron metal center and point to significant interactions within the second coordination sphere. One such interaction involves Tyr325 in human phenylalanine hydroxylase (hPAH), which forms a hydrogen-bonding network with an aqua ligand on iron and the pterin cofactor. The full-length tetramer (1-452) and truncated dimer (117-424) Tyr325Phe hPAH mutant enzymes showed similar kinetics, thermal stabilities, and oligomerization profiles as their corresponding wild-type proteins. The possibility of in vivo posttranslational hydroxylation that would restore the activity of hPAH was examined by mass spectrometry on the trypsin digested full-length (1-452) hPAH Tyr325Phe point mutant. The amino acid tags obtained by ESI-MS/MS confirmed the presence of a Phe325 in the peptide corresponding to the doubly charged precursor ion at m/z 916.4 (L A T I F W F T V E F G L C K), and its hydroxylated counterpart in the peptide corresponding to the m/z 924.4 (L A T I F-OH W F T V E F G L C K) byproduct ion series comprising the fragments y(5)-y(12). Furthermore, the point mutation Tyr325Ala resulted in an enzyme that was totally inactive and did not display any evidence of hydroxylation. These results demonstrate the importance of Tyr325 for proper conformation of the active site, substrate binding, and catalysis. The rescue of the Tyr325Phe mutant in hPAH via self-hydroxylation presents a novel example of oxidative repair on the molecular level.     

Temperature-dependent spin crossover in neuronal nitric oxide synthase bound with the heme-coordinating thioether inhibitors

A series of L-arginine analogue nitric oxide synthase inhibitors with a thioether tail have been shown to form an Fe-S thioether interaction as evidenced by continuous electron density between the Fe and S atoms. Even so, the Fe-S thioether interaction was found to be far less important for inhibitor binding than the hydrophobic interactions between the alkyl group in the thioether tail and surrounding protein (Martell et al. J. Am. Chem. Soc. 2010 , 132 , 798). However, among the few thioether inhibitors that showed Fe-S thioether interaction in crystal structures, variations in spin state (high-spin or low-spin) were observed dependent upon the heme iron oxidation state and temperature. Since modern synchrotron X-ray data collection is typically carried out at cryogenic temperatures, we reasoned that some of the discrepancies between cryo-crystal structures and room-temperature UV-visible spectroscopy could be the result of temperature-dependent spin-state changes. We, therefore, have characterized some of these neuronal nitric oxide synthase (nNOS)-thioether inhibitor complexes in both crystal and solution using EPR and UV-visible absorption spectrometry as a function of temperature and the heme iron redox state. We found that some thioether inhibitors switch from high to low spin at lower temperatures similar to the "spin crossover" phenomenon observed in many transition metal complexes.     

Protein Unfolding: 1H‐NMR Studies of Paramagnetic Ferricytochrome c‐550 from Horse Heart

Electronic transfer protein cytochrome c‐550 from horse heart is studied in the unfolded state by means of paramagnetic ¹H NMR. The protein contains 104 aminoacid residues and a heme group with low spin Fe^III ion in the oxidized form of protein. The global secondary structure is of the α‐helix type as occurs in the case of very other cytochromes c investigated such as cyt c‐550 from Thiobacillus versutus or cyt c‐551 from Pseudomonas aeruginosa. We have studied the coordination characteristic and electronic properties of heme iron horse heart ferricytochrome c‐550 at increasing denaturing conditions (up to 3.1 M GuHCl and 288‐323 K). The ¹H T₁ values of the signals were measured and some resonance assignments made based on EXSY experiments. The electronic structure of the iron(III) is discussed on the basis of the temperature dependence of the isotropic shifts and relaxation times. These results show that it is produced a change of spin, from low‐spin iron(III) (²T₂, S=1/2) in the folded state to high‐spin iron(III) (⁶A₁, S=5/2) in the unfolded state. It seems to be possible that in the opened structure the ferricyt c‐550 loses one axial ligand (His/‐) appearing the spin transition.     

Effect of a negative charge on the screening of the active site of horseradish peroxidase

The F143E mutant form of the recombinant horseradish peroxidase was reactivated fromE. coli inclusion bodies. The mutation inhibits heme entrapment and results in a decrease in the catalytic activity, mainly affecting the stage of the oxidation of a donor substrate (ABTS, iodide). An increase in stability of the mutant form obtained under radiation inactivation over that of the wild-type recombinant enzyme was observed. The data obtained confirms the proposed location of Phel43 at the entrance of the active center, hence its replacement by the negatively charged glutamic acid residue retards heme entrapment and substrate binding, thus protecting the active center of the enzyme against the radicals generated by radiolysis.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 371–374, February, 1995.     

Ultrafast heme dynamics in ferrous versus ferric cytochrome c studied by time-resolved resonance Raman and transient absorption spectroscopy

Cytochrome c (Cyt c) is a heme protein involved in electron transfer and also in apoptosis. Its heme iron is bisaxially ligated to histidine and methionine side chains and both ferric and ferrous redox states are physiologically relevant, as well as a ligand exchange between internal residue and external diatomic molecule. The photodissociation of internal axial ligand was observed for several ferrous heme proteins including Cyt c, but no time-resolved studies have been reported on ferric Cyt c. To investigate how the oxidation state of the heme influences the primary photoprocesses, we performed a comprehensive comparative study on horse heart Cyt c by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We found that in ferric Cyt c, in contrast to ferrous Cyt c, the photodissociation of an internal ligand does not take place, and relaxation dynamics is dominated by vibrational cooling in the ground electronic state of the heme. The intermolecular vibrational energy transfer was found to proceed in a single phase with a temperature decay of approximately 7 ps in both ferric and ferrous Cyt c. For ferrous Cyt c, the instantaneous photodissociation of the methionine side chain from the heme iron is the dominant event, and its rebinding proceeds in two phases, with time constants of approximately 5 and approximately 16 ps. A mechanism of this process is discussed, and the difference in photoinduced coordination behavior between ferric and ferrous Cyt c is explained by an involvement of the excited electronic state coupled with conformational relaxation of the heme.     

The H93G myoglobin cavity mutant as a versatile template for modeling heme proteins: ferrous, ferric, and ferryl mixed-ligand complexes with imidazole in the cavity

One of the difficulties in preparing accurate ambient-temperature model complexes for heme proteins, particularly in the ferric state, has been the generation of mixed-ligand adducts: complexes with different ligands on either side of the heme. The difference in the accessibility of the two sides of the heme in the H93G cavity mutant of myoglobin (Mb) provides a potential general solution to this problem. To demonstrate the versatility of H93G Mb for the preparation of heme protein models, numerous mixed-ligand adducts of ferrous, ferric, and ferryl imidazole-ligated H93G (H93G(Im) Mb) have been prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analogous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spectroscopically resembles wild-type ferric Mb as expected for a complex containing a single imidazole in the proximal cavity and water bound on the distal side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge neutral (imidazole) or anionic (cyanide and azide), results in formation of six-coordinate low-spin complexes with MCD characteristics similar to those of parallel derivatives of wild-type ferric Mb. Reduction of ferric H93G(Im) Mb and subsequent exposure to either CO, NO, or O2 produces ferrous complexes (deoxy, CO, NO, and O2) that consistently exhibit MCD spectra similar to the analogous ferrous species of wild-type ferrous Mb. Most interestingly, reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are essentially identical to those of oxoferryl wild-type Mb. The generation of such a wide array of mixed-ligand heme complexes demonstrates the efficacy of the H93G Mb cavity mutant as a template for the preparation of heme protein model complexes.