Coordination geometry of heme, in lactoperoxidase. pH-Dependent <SUP>1</SUP>H relaxivity and optical spectral studies

Molar relaxivity of water proton in lactoperoxidase solution was studied as a function of pH in the range of 2-13 by spin-lattice relaxation time measurements on a Bruker AM 500 MHz nuclear magnetic resonance (NMR) spectrometer. It was shown by comparison with the molar relaxivities of met myoglobin (Mb) and horseradish peroxidase (HRP) solutions that the sixth coordination position of the heme pocket in lactoperoxidase (LPO) is vacant. Distance of the water proton in the heme pocket from ferric ion was deduced to be 2.7, 3.6 and 4.3 A for Mb, HRP, and LPO, respectively. Acid-alkaline transition for met myoglobin, horseradish peroxidase, and lactoperoxidase determined from the pH dependence of changes in the Soret absorptions were found to be characterized by pK of 8.8, 10.9, and 12.1, respectively. Proton NMR of LPO at pH = 12.2 was found to have single broad resonance considerably upfield shifted as compared to that of LPO at neutral pH. By comparison with the proton NMR of HRP and Mb at pH greater than their respective pK of acid-alkaline transition, the upfield shifted proton resonance of LPO at pH = 12.2 was assigned to be due to low-spin LPO

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH&#8804;4 and that the oxidation does not take place at pH&#8805;6. Since thiocyanate does not bind to HRP at pH&#8805;6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to PO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated

Binding of heme to human serum albumin: Steady-state fluorescence, circular dichroism and optical difference spectroscopic studies

7-12The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at ~397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA]=1. Titration of HSA with heme was followed by ODS and the dissociation constant KD = (4.0±1.0)×10⁻⁵ M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protei

13C Nuclear magnetic resonance studies of binding of thiocyanate to lactoperoxidase and horseradish peroxidase heme enzymes

301-311Interaction of thiocyanate with lactoperoxidase (LPO) and horseradish peroxidase (HRP) has been investigated by relaxation rate measurements (at 125.77 MHz) of 13C resonance of thiocyanate carbon. The apparent dissociation constant (KD) for thiocyanate binding to LPO at pH = 6.1 and to HRP at pH = 4.0 has been deduced to be 85 mM and 160 mM respectively from the relaxation rate measurements. The pH dependence of KD and 13C resonance line-width of thiocyanate has been used to calculate pKa value of amino acid residue on these enzymes where the thiocyanate is shown to be binding. From the pH dependence of KD and 13C resonance line-width, it is observed that thiocyanate binds to LPO and HRP only under acidic conditions (pH pH pKa = 6.1 for LPO and 4.0 for HRP. The pH dependence of 13C resonance line-width of thiocyanate as well as KD have been quantitatively analysed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. KD for thiocyanate binding to the enzyme has also been evaluated in the presence of excess of exogenous substrates such as resorcinol, cyanide and iodide. The presence of cyanide (which binds to heme iron of enzyme at sixth coordination position) does not have any effect on the binding of thiocyanate, indicating that binding site of thiocyanate ion is located away from the ferric centre of these enzymes. The presence of resorcinol, has significant effect on KD for binding of thiocyanate to LPO but it has no effect on thiocyanate binding to HRP. The K D in the presence of iodide however shows that iodide competes with thiocyanate for binding at the same site in both the cases. Distance of the carbon atom of bound thiocyanate ion from ferric centre has been deduced from the 13C-TJ measurements and is found to be 8.4 Ǻ and 8.0 Ǻ for LPO and HRP respectively. This distance remains unchanged by the presence of cyanide ion at the sixth coordination site of the heme iron of these enzymes. Similarity in the mode of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2, like that of 1- by H2O2 may also proceed via two-electron transfer pathway under acidic conditions

Binding of thiocyanate to lactoperoxidase: proton and nitrogen-15 nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 &#177; 5, 173 &#177; 20, and 83 &#177; 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 &#177; 0.2 A from the 15N T1 measurements

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicate that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 &#216;A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a <SUP>15</SUP>N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 &#197;, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 &#197;. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion