Spectral Studies of Iron Coordination in Hemeprotein Complexes: Difference Spectroscopy below 250 mμ

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.     

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.     

Magnetic and spectroscopic probes for FeOFe linkages in hemin systems.

Magnetic and spectroscopic properties of mu-oxo-bis-hemins from natural and structurally related porphyrins were investigated as probes for ascertaining the presence or absence of FeIII-O-FeIII linkages between hemin moieties of hemeproteins. Magnetic susceptibilities of solids from 2.2 to 293 degrees K were investigated. The data fit the temperature variations expected for a pair of antiferromagnetically coupled S = 5/2, iron (III) porphyrins with J values of 175, 190, 195, 205, and 210 degrees K for deuterohemins with hydrogen, vinyl, 2'-ethoxycarbonylcyclopropyl, acetyl, propionyl, and ethyl 2,4-substituents, respectively. This magnetic character is reflected in PMR spectra that exhibit resonances with far less broadening and paramagnetic shift than is the case for monomeric high-spin hemins. Only impurities are seen in EPR spectra, which serve effectively in monitoring the magnetic purity of preparations. An infrared active asymmetric stretching frequency characteristic of the FeOFe linkage can be identified by substitution of 160 by 180. Electronic spectra are highly characteristic with poorly resolved absorption bands. The substituents on the porphyrin ring exert significant, but usually not large, electronic and steric effects on these properties. Solvent effects were relatively small and no firm evidence for binding of ligands trans to bridging oxygen was found. The uniqueness of these physical properties and their low sensitivity to changes in porphyrin structure or medium facilitates the identification of mu-oxo linkage in hemins or oxidized hemeproteins.     

Coupling of dihydroriboflavin oxidation to the formation of the higher valence states of hemeproteins.

The reactions between hydrogen peroxide and hemeproteins have been coupled to the oxidation of dihydroriboflavin so as to provide a simple method for measuring the rate constant of hemeprotein peroxidation. Dihydroriboflavin rapidly reduces the higher oxidation states of iron and the hydroxy radicals which are the products of the hemeprotein/hydrogen peroxide reaction. The rapid reduction of these highly reactive compounds prevents the hemeproteins from undergoing irreversible chemical modifications and thus allows the kinetics of peroxidation to be studied. The rate constants at pH 7.2 and 23 degrees C for the peroxidation of horseradish peroxidase, myoglobin, and ferrocytochrome c are found to be 6.2 x 10(6), 7.5 x 10(4), and 8 x 10(3)M-1s-1, respectively. These studies suggest that reduced riboflavin might efficiently protect cells from oxidative damage such as that occurring in inflammation and reperfusion injury.     

Binding mode of indole to horseradish peroxidase

The binding of indole to both horseradish peroxidase and its cyanide complex can be detected by difference spectra in the Soret region. Indole and cyanide binding are not competitive processes. The effect of indole on the binding rate constants between horseradish peroxidase and cyanide and compound I formation reactions between horseradish peroxidase and hydrogen peroxide or m-chloroperbenzoic acid was studied by the stopped-flow method. In all cases the rate constants of the indole-peroxidase complex with the ligand or substrates were smaller than those of free peroxidase. Since the m-chloroperbenzoic acid reaction has been shown to approach a diffusion-controlled rate, the effect of indole binding on the rate constant for compound I formation using this peracid was analyzed semiquantitatively using theoretical equations for a diffusion-controlled rate process with a capture-window active site model. The effect of indole binding on the diffusion-controlled rate constant could be explained by a decrease in the radius of the capture-window active site.     

A study of ligand binding to spleen myeloperoxidase

The ligand binding properties of spleen myeloperoxidase, a peroxidase formerly called "the spleen green hemeprotein", were studied as functions of temperature and pH, using chloride and cyanide as exogenous ligands. Ligand binding is influenced by a proton dissociable group with a pKa of 4. The protonated, uncharged form of cyanide binds to the unprotonated form of the enzyme, while chloride ion binds to the enzyme when this group is protonated. In both cyanide and chloride binding, the pH-dependent change in the apparent ligand affinity is due to a change in the apparent association rate with pH. The proton dissociable group on the enzyme involved in ligand binding has a delta H value of about 8 kcal . mol-1. The present results suggest that this ionizable group is the imidazole group of a histidine residue located near the ligand binding site.     

Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase

The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase.     

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.     

Structural characterization of lactoperoxidase in the heme environment by proton NMR spectroscopy

The heme environmental structures of lactoperoxidase (LP) have been studied by the use of hyperfine-shifted proton NMR and optical absorption spectra. The NMR spectra of the enzyme in native and cyanide forms in H2O indicated that the fifth ligand of the heme iron is the histidyl imidazole with an anionic character and that the sixth coordination site is possibly vacant. These structural characteristics are quite similar to those of horseradish peroxidase (HRP), suggesting that these may be prerequisite to peroxidase activity. The pH dependences of the spectra of LP in cyanide and azide forms showed the presence of two ionizable groups with pK values of 6 and 7.4 in the heme vicinity, which is consistent with the kinetic results. The group with pK = 7.4 is associated with azide binding to LP in a slow NMR exchange limit, which is in contrast to the fast entry of azide to HRP.     

Iron coordination in photosystem II: interaction between bicarbonate and the QB pocket studied by Fourier transform infrared spectroscopy

The non heme iron environment of photosystem II is studied by light-induced infrared spectroscopy. A conclusion of previous work [Hienerwadel, R., and Berthomieu, C. (1995) Biochemistry 34, 16288-16297] is that bicarbonate is a bidendate ligand of the reduced iron and a monodentate ligand in the Fe(3+) state. In this work, the effects of bicarbonate replacement with lactate, glycolate, and glyoxylate, and of o-phenanthroline binding are investigated to determine the specific interactions of bicarbonate with the protein. Fe(2+)/Fe(3+) FTIR spectra recorded with (12)C- and (13)C(1)-labeled lactate indicate that lactate displaces bicarbonate by direct binding to the iron through one carboxylate oxygen and the hydroxyl group in both the Fe(2+) and Fe(3+) states. This different binding mode with respect to bicarbonate could explain the lower midpoint of the iron couple observed in the presence of this anion [Deligiannakis, Y., Petrouleas, V., and Diner, B. A. (1994) Biochim. Biophys. Acta 1188, 260-270]. In agreement with the -60 mV/pH unit dependence of the iron midpoint potential in the presence of bicarbonate, the proton release upon iron oxidation by photosystem II is directly measured to 0.95 +/- 0.05 by the comparison of infrared signals of phosphate buffer and ferrocyanide modes. This accurate method may be applied to the study of other redox reactions in proteins. The pH dependence of the iron couple is proposed to reflect the deprotonation of D1His215, a putative iron ligand located at the Q(B) pocket, since the signal at 1094 cm(-1) assigned to the nu(C-N) mode of a histidinate ligand in the Fe(3+) state is not observed in the presence of o-phenanthroline. Specific regulation of the pK(a) of D1His215 by bicarbonate is inferred from the absence of the band at 1094 cm(-1) in Fe(2+)/Fe(3+) spectra recorded with glycolate, glyoxylate, or lactate. A broad positive continuum, maximum at approximately 2550 cm(-1), observed in the presence of bicarbonate, but absent with o-phenanthroline or lactate, glycolate, and glyoxylate, indicates a hydrogen bond network from the non heme iron toward the Q(B) pocket involving bicarbonate and His D1-215. Proton release of about 1, measured upon iron oxidation at pH 6 with the latter anions, points to a proton release mechanism different from that involved in the presence of bicarbonate.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Proton NMR study of the cyanide metmyoglobin reconstituted with meso-tetraalkylhemins. Dynamic free rotation of the synthetic hemins in the heme pocket

Incorporation of the three synthetic hemins, Fe(III) meso-tetraalkylporphyrins with the methyl, ethyl, or n-propyl groups, into apomyoglobin was followed by spectrophotometry, and the stoichiometric complex formation was confirmed. The reconstituted myoglobins bind with an equimolar amount of cyanide to exhibit visible absorption peaks at 419, 570, and 608 nm. The spectral feature was independent of the cyanide concentrations. Proton NMR spectra of the cyanide complexes resolved the pyrrole-proton signals of the hemins in a -5 to -15-ppm region, which is comparable with that of the corresponding signals of deuterohemin-containing low-spin methemoproteins. These spectral observations indicate the presence of the NC-Fe-N(His-F8) structure in the presently reconstituted cyanide metmyoglobins. The pyrrole-proton NMR signals of the hemins in cyanide metmyoglobins appeared as a singlet, doublet, or quartet for the methyl, ethyl, or n-propyl hemin complexes, respectively. The systematic NMR spectral changes suggest the dynamic free rotation of the alkylhemins about the Fe-N(His-F8) bond. Temperature-dependent NMR spectral transition of the meso-tetraethylhemin-reconstituted myoglobin was consistent with thermally regulated dynamic free rotation of the hemin in the myoglobin heme pocket.     

Spectroscopic properties of ferrous heme complexes of sterically hindered ligands.

Mesoheme IX complexes of sterically hindered ligands 2-methylimidazole, tert-butylamine and 2-methylpyridine in aqueous glycerol solutions are characterized by broad visible absorption spectra at ambient temperature exhibiting close similarities to high-spin ferrous hemeproteins. Spectrophotometric titrations of mesoheme IX with these ligands indicate well-defined equilibria for 2-methylimidazole and tert-butylamine corresponding to the formation of penta-coordinate strong-field ligand complexes. Variable temperature spectra of these complexes from ambient to 77 degrees K exhibit a change to hemochrome spectra characteristic of the low-spin unhindered ligand complexes. Corresponding changes in the visible spectra are not observed for the high-spin hemeproteins deoxymyoglobin, horse-radish peroxidase and cytochrome ?. The appropriate utilization of these hindered ligand heme complexes as model systems for high-spin ferrous hemeproteins has been discussed.     

Mechanisms of cytochrome P450 and peroxidase-catalyzed xenobiotic metabolism.

The cytochrome P450 enzyme systems catalyze the metabolism of a wide variety of naturally occurring and foreign compounds by reactions requiring NADPH and O2. Cytochrome P450 also catalyzes peroxide-dependent hydroxylation of substrates in the absence of NADPH and O2. Peroxidases such as chloroperoxidase and horseradish peroxidase catalyze peroxide-dependent reactions similar to those catalyzed by cytochrome P450. The kinetic and chemical mechanisms of the NADPH and O2-supported dealkylation reactions catalyzed by P450 have been investigated and compared with those catalyzed by P450 and peroxidases when the reactions are supported by peroxides. Detailed kinetic studies demonstrated that chloroperoxidase- and horseradish peroxidase-catalyzed N-demethylations proceed by a Ping Pong Bi Bi mechanism whereas P450-catalyzed O-dealkylations proceed by sequential mechanisms. Intramolecular isotope effect studies demonstrated that N-demethylations catalyzed by P450s and peroxidases proceed by different mechanisms. Most hemeproteins investigated catalyzed these reactions via abstraction of an alpha-carbon hydrogen whereas reactions catalyzed by P-450 and chloroperoxidase proceeded via an initial one-electron oxidation followed by alpha-carbon deprotonation. 18O-Labeling studies of the metabolism of NMC also demonstrated differences between the peroxidases and P450s. Because the hemeprotein prosthetic groups of P450, chloroperoxidase, and horseradish peroxidase are identical, the differences in the catalytic mechanisms result from differences in the environments provided by the proteins for the heme active site. It is suggested that the axial heme-iron thiolate moiety in P450 and chloroperoxidase may play a critical role in determining the mechanism of N-demethylation reactions catalyzed by these proteins.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.     

Bovine Carbonyl Lactoperoxidase Structure at 2.0? Resolution and Infrared Spectra as a Function of pH

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I^? into antimicrobials and small aromatic organics after being itself oxidized by H₂O₂. LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO?CLPO at 2.0? resolution and infrared (IR) spectra of the iron-bound CO stretch from pH?3 to 8.8?at 1 cm^?1 resolution. The observed Fe?CC?CO bond angle of 132° is more acute than the electronically related Fe(III), CN?CLPO with a Fe?CC?CN angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN?CLPO. The IR spectra of CO?CLPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ?? 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH?7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.     

Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.

Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation.     

Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.