The transformation of chlorophenols by lactoperoxidase.

The lactoperoxidase-catalyzed transformations of penta-,2,3,4,6-tetra-, 2,4,6-tri-, 2,4-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol:H2O2 ranged from 1:1 for the tri- and tetrachlorophenol at pH7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (v0) was only slightly influenced by changes in [H2O2] greater then 5 microns. v0 responded to [chlorophenol] according to the empirical expression v0=[lactoperoxidase] . (k1[chlorophenol] + k2[chlorophenol]2). The constant k1 was trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 microns for H2O2 and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g of peroxidase-containing tissue, possibly with deposition of the products.

The vibrational bands of carbon monoxide bound to hemes or metal surfaces.

Infrared spectra of imidazole carbonyl complexes of 2,4-substituted hemes are presented. An increased CO stretch frequency is accompanied by a lowered FeC vibrational energy. Hartree-Fock-Slater electron structure calculations discern pi and sigma contributions to the observed shifts of vibrational energies. We conclude that an enhanced electron availability manifests itself as a lowered ferric/ferrous reduction potential, increased filling of the 2 pi orbital of liganded CO which in turn reduces nu CO and increases nu Fec, and increased basicity of the liganded CO. Analogies between CO liganded to heme and CO adsorbed onto metal surfaces are discussed.

Fast heme release from inactive proteins.

Kinetics for the release of the prosthetic group from hemoproteins is presented. Heme-protein separation is a biphasic reaction, where an initial phase is significantly faster than the dominant, slow phase. A previous communication concluded that the slow phase represents the active protein. This communication presents data for porphyrin release which shows that the initial fast phase represents an inactive form of the protein. Moreover, we suggest that the fast to slow phase ratio is a sensitive monitor of sample quality for many hemoproteins and that an extrapolation of the slow phase absorbance leads to new estimates for the true physical parameters of unperturbed proteins.

Spin and electron distributions in heme-cyanide models and hemeproteins.

Proton NMR spectra of low-spin Fe(III) cyanoprotoheme as prosthetic group in a number of proteins are presented. The diagonally positioned 1-, 5- and 3-, 8-methyl groups obey shifts proportional to the Fe(III)/(II) reduction potential Em7, which indicates a pseudo-contact interaction. The correlation with Em7 is understandable if one postulates an enhanced rhombic distortion, dominating the Fe-methyl dipolar interactions. Hartree-Fock-Slater quantum chemical calculations show no significant changes of spin density as a function of the Fe-L5 distance, except at the iron atom and predominantly in the 3dxz and 3dyz orbitals. 4p orbitals, on the other hand, uphold most of the changes of electron density. We also observe a principal difference in the amino acid sequences in the heme-accommodating pocket of oxygen carriers and two-electron transmitters.

Ab initio calculations of electron distributions in heme-CO models.

We have, by the use of ab initio calculations, found a back-bonding state of pi symmetry close to the Fermi level for CO bound to FeN5C14. We thus find it likely that small shifts of the redox potential magnitude of EF - EV magnitude of will cause relatively large changes of the CO vibrational frequency. The separation of Fe 3d orbitals in our heme model is found to agree with what is predicted by ligand field theory for Oh symmetry. This paper presents nonrelativistic Hartree-Fock-Slater calculations of the 5 sigma bonding and 2 pi back-bonding between CO and Fe. The effects of up to 19 additional atoms are discussed for models of heme (COFe to COFeN5C14). The filled back-bonding state is found to be strongly influenced by second nearest neighbor atoms. By use of symmetry orbitals we have resolved the Fe 3d orbitals into the T2g and Eg representations of the Oh point group and find the former states to be occupied whereas the latter are unoccupied. The difference in occupancy is reduced when the CO ligand is removed which also causes an increased density of states at the Fermi level, i.e., the highest occupied and lowest unoccupied orbitals. Possible correlations between our data and experimental results are discussed for heme proteins as well as for metal surfaces.

A 13C-NMR study of mutant hemoglobins with altered oxygen affinity.

The 13CO-NMR spectra of carbonylhemoglobins Saint Mandé (beta 102Asn----Tyr), Malmö (beta 97His----Gln), Hôtel Dieu (beta 99Asp----Gly) and Ao have been determined. The positions of the 13CO resonances for hemoglobins Ao, Malmö and Hôtel Dieu were similar indicating similar ligand environments for all. The 13CO resonance for the beta-subunit of Saint Mandé was upfield-shifted compared to the others. This is evidence that structural changes at the beta 102 position directly affect iron-ligand bonding as well as quaternary structure.

Mutant hemoglobin stability depends upon location and nature of single point mutation.

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.

Bovine carbonyl lactoperoxidase structure at 2.0Å 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 H(2)O(2). 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-LPO at 2.0Å 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-C-O bond angle of 132° is more acute than the electronically related Fe(III), CN-LPO with a Fe-C-N 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-LPO. The IR spectra of CO-LPO 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.

Peroxidases: past and present.

The history of peroxidases spans nearly two centuries. Our knowledge has developed from early phenomenological observations of the colored products of peroxidase-catalyzed reactions, to our present understanding of many of the steps in the complex peroxidation reaction mechanism. Peroxidases are ubiquitous in plant and animal tissues and occur in diverse structural forms. Collectively, they are able to catalyze the hydroperoxide oxidation of many different kinds of organic and inorganic compounds. In spite of the great diversity of structures and functions, mechanisms of heme-containing peroxidases have several common features: (i) the transfer of the oxidizing equivalents of the hydroperoxidase to the enzyme to form Compound I, (ii) the reduction of Compound I by the transfer of electrons from donor molecules, (iii) the inactivation of Compound I by excess hydroperoxide. Rate constants for these and other steps in the peroxidation mechanism, as well as redox potentials, have been reported for many peroxidases. The molecular basis for the donor specificity of peroxidases has not yet been elucidated. Today, much interest is directed towards the biological functions of peroxidases and their reaction products.

Peroxidase activity and thiocyanate accumulation in salivary glands.

Salivary glands with high, low, or no peroxidase activity do not differ in [S14CN-] after the i.v. injection of KS14CN, nor do the glands differ from blood and muscle in [S14CN-]. The content of SCN- in a salivary gland does not mirror the gland's participation in the peroxidase-mediated antimicrobial mechanism.

The reduction potential of lactoperoxidase.

The reduction potential of Fe(III)/Fe(II) lactoperoxidase has been determined. Optical determinations of equilibria with 2-methyl-3-hydroxy-1,4-naphthoquinone as indicator and prereduced 9,10-anthraquinone-2-sulfonate as reducing agent gave Em,7.0 = -191 +/- 2 mV. Potentiometric determinations with 9,10-anthraquinone-2-sulfonate as mediator and, in the reduced form as reducing agent, gave Em,7.0 = -188 +/- 1 mV. Addition of 0.5% N-cetyl-N,N,N-trimethyl-ammonium bromide, and using dithionite as reducing agent, gave Em,7.0 = -183 mV and -179 mV with 9,10-anthraquinone-2-sulfonate and 9,10-anthraquinone-2,6-disulfonate, respectively, as mediators.

Equilibria between horseradish peroxidase and aromatic donors.

Equilibria between horseradish peroxidase and aromatic hydrogen donors have been analyzed spectrophotometrically and potentiometrically. The donors alter the peroxidase spectrum slightly but reproducibly with changes of two types. Donors of the two groups compete for the same binding site with no systematic difference in affinity for the enzyme. Donors with one aromatic ring are fairly loosely ligated, Kd3-25 mM, but enlargement, or extension of the pi-electron system, increases the affinity. A negative change in entropy and a large negative change in enthalpy upon binding indicates a specific donor-enzyme interaction, and the retention of the peroxidase by phenyl- but not by octyl-Sepharose points at the involvement of aromatic amino acid(s) in the ligation of an aromatic donor. Substitution of the hematin vinyl groups by ethyl or acetyl groups does not affect Kd of the peroxidase-donor complex. Reduction of the iron atom to Fe(II), or its removal, influences Kd only modestly. The fluorescence of the protoporphyrin-apoprotein HRP C2 associate is not quenched by donors from either group. These observations are in accord with NMR and other data from the literature and point at a ligation of the donor only to the protein moiety. Our results do not support the assumption of an Fe(III) H2O...donor hydrogen bond. The energy balance in the four-membered system free and donor-bound peroxidase Fe(III)/(II) has been analyzed. The model donors used in the present study modulate the redox properties only slightly. Plant peroxidases in situ may be donor-bound to a large extent.

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing.

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.