Comparison of the heme electron state of reduced cytochrome P450 and P420 in equilibrium and non-equilibrium protein conformations. The nature of the protein heme ligand

Magnetic circular dichroism (MCD) spectra of reduced cytochromes P450 and P420 in equilibrium and non-equilibrium protein conformations are compared at 4.2 K for the 350-800 spectral region. Non-equilibrium forms have been produced by photolysis of CO-complexes at 4.2 K. The differences between MCD spectra of proteins in equilibrium and non-equilibrium conformations, in particular for the visible region, show clearly the structural changes in the heme iron coordination sphere to occur on ligand binding. The comparison of the Soret MCD spectra of reduced proteins in their equilibrium and non-equilibrium forms with those of other high-spin ferrous hemoproteins suggest that mercaptide (RS-) is the protein ligand of the heme iron in reduced P450, as well as in its CO-complex, and that imidazole of histidine is the fifth ligand of the iron both in reduced P420 and its CO-complex. The thermal recombination of the photoproducts with CO have been studied. When temperature rises from 4.2 to 77 K for two hours both proteins have similar temperature characteristics during the recombination processes. The recombination begins at T approximately equal to 10 K and is completed at approximately equal to 50 K. The temperature at which half of the total photolyzed molecules are restored to the CO-form is equal to 25 K. For products of photolysis of CO-complexes of myoglobin and hemoglobin under the same heating conditions these temperatures are equal to 35 and 23 K respectively. Thus, the photoproducts of P450, P420 and hemoglobin have similar parameters of low-temperature recombination and the kinetics of this process is faster than for photodissociated myoglobin.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

Magnetic circular dichroism studies of myoglobin,hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q ₀₀- and Q_v-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q ₀₀-band to the A-term in the Q _v-band. The evidences are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the hemoglobin-like MCD, while the alkaline ferroperoxidase is characterized by the myoglobin-like MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoproteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-ion charge-transfer electronic transition which may be assigned as b() 3d. This charge-transfer band is strongly overlapped with usual B( - *) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.     

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Spectral evidence for sub-picosecond iron displacement after ligand detachment from hemoproteins by femtosecond light pulses.

We have measured spectral and kinetic differences in protoheme, sperm whale or horse heart myoglobin and human hemoglobin following photodissociation induced by optical pulses of 80 fs duration. Full ligation was performed with oxygen or carbon monoxide. Femtosecond kinetics and transient difference spectra revealed the appearance of a deoxy species with tau approximately equal to 250-300 fs. The transient deoxy species in myoglobin and hemoglobin evidenced a 3-4 nm red shift of their delta A spectra compared with the equilibrium delta A spectrum. This shift was not observed after photodissociation of the carbon monoxide liganded protoheme. We proposed that the 250 fs time constant corresponding to the appearance of the deoxy-like species is related to the displacement of the ferrous iron out of the heme plane. Consequently, the small red shift of the delta A spectra observed in photodissociated hemoproteins may be tentatively attributed to changes in the vibrational modes of either the proximal histidine-Fe2+ bond and/or of the N4 porph-Fe-N epsilon His (F8) bent.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Contribution of Protein Conformation to Heme Stereochemistry and Reactivity. Low-Temperature Magnetic Circular Dichroism Data

Abstract

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O₂ complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N_epslion(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those inthe protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Absorption and magnetic circular dichroism spectra of hemoproteins in nonequilibrium states. V. Cytochrome P450 and its substrate complex

It was shown that ferrocytochrome P450 forms a nonequilibrium state if ferrocytochrome P450 and its complexes are reduced in freezed water-glycerol solutions by thermolysed electrons, arising during gamma-radiolysis of the matrix at 77 degrees K. Unlike the equilibrium form of ferrocytochrome P450 with the heme iron at the high-spin state the reduced nonequilibrium form of the protein contains the heme iron at a low-spin state. The absorption spectrum of ferrocytochrome P450 in the nonequilibrium state is characterized by alpha and beta-bands at 562 and 534 nm, respectively, whereas the magnetic circular dichroism spectra exhibit type A effect at 562 nm. Upon temperature increasing the nonequilibrium state is relaxed to the equilibrium one. Type 1 substrates had practically no influence on the spectral characteristic of the nonequilibrium form of ferrocytochrome P450. Binding of type 2 substrates results in an essential decrease of the intensity ratio of the alpha- and beta-bands (A alpha/A beta) and is accompanied by a red-shift of the alpha-band and corresponding magnetic circular dichroism effect. It was shown that mercaptoethanol complex of hemoglobin, formed by reduction at 77 degrees K is spectrally similar to the nonequilibrium ferrocytochrome P450 complex with type 2 substrates. From analysis of experimental data one can conclude that (i) the ligand environment of heme iron in oxidased and reduced cytochrome P450 are different; (ii) the sixth axial ligand of the heme iron in the oxidised protein is probably a water molecule (OH-) attached by a hydrogen bond to the neighbouring histidine. It is assumed that a similar nonequilibrium form of cytochrome P450 can be formed in physiological conditions.     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Expression,purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.     

On the use of iron octa-alkylporphyrins as models for protoporphyrin IX-containing heme systems in studies employing magnetic circular dichroism spectroscopy.

The magnetic circular dichroism (MCD) properties of numerous oxidation and ligation state derivatives of myoglobin and horseradish peroxidase reconstituted with an iron octa-alkylporphyrin (mesoheme IX) have been investigated in order to establish the utility of such porphyrins as models for protoporphyrin IX-containing systems. The MCD spectra of the mesoheme-reconstituted proteins are blue-shifted (4-12 nm) and are somewhat more intense (1.5-2.5 fold) when compared to the spectra of analogous derivatives of native myoglobin and horseradish peroxidase. However, the spectral band patterns of the mesoheme-reconstituted proteins closely resemble those of the native proteins in essentially all cases. These data demonstrate that octa-alkylporphyrins can be productively used as models for protoporphyrin IX in studies of heme proteins with MCD spectroscopy.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)