Magnetic resonance spectroscopy, calcium content, and anion coordination studies of bovine and goat lactoperoxidase.

Bovine lactoperoxidase from two purebred strains and a commercial source as well as lactoperoxidase isolated from Alpine goat milk were examined by proton NMR spectroscopy for structural comparison of the heme site. Hyperfine shifted proton NMR spectra for both the native enzymes and cyanide complexes were equivalent for the protein obtained from the four separate sources. Activity assays (guaiacol and iodide ion oxidations) were also employed to compare the enzyme from various sources. Bovine lactoperoxidase was shown to contain 1.5 +/- 0.1 calcium ions per heme unit. Lactoperoxidase complexes with nitrite ion and thiocyanate ion were characterized for comparison with the cyanide complex. The nitrite complex exhibits a proton NMR hyperfine shift pattern at ambient temperature consistent with a low-spin ferric formulation. Interaction of lactoperoxidase with thiocyanate ion was monitored by NMR and EPR spectroscopy. Proton NMR spectra of lactoperoxidase in the presence of excess thiocyanate ion illustrated the retention of a high-spin ferric configuration consistent with predominant binding of the physiological thiocyanate substrate at a non-heme site at room temperature. However, EPR spectroscopy at cryogenic temperatures revealed the existence of a low-spin lactoperoxidase thiocyanate complex. This result may be explained by low-affinity ambient temperature thiocyanate heme binding that is greatly enhanced at liquid helium temperature.

Response regulation in bacterial chemotaxis.

The signal transduction system that mediates bacterial chemotaxis allows cells to modulate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY.

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].

Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors.

Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that receive phosphoryl groups from the histidine kinase, CheA. Here we show that the response regulators catalyze their own phosphorylation in that both CheY and CheB can be phosphorylated in the complete absence of any auxiliary protein. Both CheY and CheB use the N-phosphoryl group in phosphoramidate (NH2PO3(2-)) as a phospho-donor. This enzymatic activity probably reflects the general ability of response regulators to accept phosphoryl groups from phosphohistidines in their associated kinases. It provides a general method for the study of activated response regulators in the absence of kinase proteins. CheY can also use intermediary metabolites such as acetyl phosphate and carbamoyl phosphate as phospho-donors. These reactions may provide a mechanism to modulate cell behavior in response to altered metabolic states.

Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis.

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY.

Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis.

Signal transduction in bacterial chemotaxis involves transfer of a phosphoryl group between the cytoplasmic proteins CheA and CheY. In addition to the established metal ion requirement for autophosphorylation of CheA, divalent magnesium ions are necessary for the transfer of phosphate from CheA to CheY. The work described here demonstrates via fluorescence studies that CheY contains a magnesium ion binding site. This site is a strong candidate for the metal ion site required to facilitate phosphotransfer from phospho-CheA to CheY. The diminished magnesium ion interaction with CheY mutant D13N and the lack of metal ion binding to D57N along with significant reduction in phosphotransfer to these two mutants are in direct contrast to the behavior of wild-type CheY. This supports the hypothesis that the acidic pocket formed by Asp13 and Asp57 is essential to metal binding and phosphotransfer activity. Metal ion is also required for the dephosphorylation reaction, raising the possibility that the phosphotransfer and hydrolysis reactions occur by a common metal-phosphoprotein transition-state intermediate. The highly conserved nature of the proposed metal ion binding site and site of phosphorylation within the large family of phosphorylated regulatory proteins that are homologous to CheY supports the hypothesis that all these proteins function by a similar catalytic mechanism.

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase, cytochrome P-450cam, and porphinatoiron(II) thiolate complexes.

The reduced forms of cytochrome P-450cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by 15N-NMR spectroscopy of the cyanide complexes of the ferric forms.

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.

Electron paramagnetic resonance spectroscopy of thyroid peroxidase.

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase.

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase.

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.

Sulfide-bridged derivatives of the binuclear iron site of hemerythrin at both met and semi-met oxidation levels.

Exposure of methemerythrin (metHr) to S2- under anaerobic conditions results in a one-electron reduction to the semi-met level and replacement of the mu-oxo bridge between the irons with a single sulfide. The sulfide bridge is maintained upon ferricyanide oxidation of semi-metsulfide to metsulfide hemerythrin and upon subsequent dithionite or S2- reduction back to the semi-met level. Chemical analyses show that metsulfideHr contains one S2- per two Fe. The single quadrupole doublet (delta = 0.50 mm/s; delta Eq = 0.99 mm/s) in the Mössbauer spectrum is consistent with a bridging sulfide geometry. The optical and resonance Raman spectra of metsulfideHr are reminiscent of the [2Fe-2S] iron-sulfur proteins. The optical spectrum exhibits multiple S2----Fe(III) charge-transfer transitions between 400 and 600 nm. The resonance Raman spectrum reveals a series of overtones and combinations of the 431-cm-1 Fe-S-Fe symmetric vibration and the 327-cm-1 asymmetric vibration. The relative energies of the symmetric and asymmetric modes are characteristic of a sulfur-bridged system with a bridge angle of approximately 80 degrees. MetsulfideHr decomposes over several hours in air and over several days in the absence of O2 to metHr and semi-metsulfideHr, respectively. Unlike metHr and semi-metHr, neither the metsulfide nor the semi-metsulfide derivatives form stable adducts with anions such as azide or cyanide. Sulfide bridging confers new properties on the binuclear iron center that are of interest to an understanding of the chemistry of hemerythrin and also of the [2Fe-2S] iron-sulfur proteins.

Semi-met oxidation level of chalcogenide derivatives of methemerythrin. Mössbauer and EPR studies.

Conclusive evidence is presented for an S = 1/2 spincoupled pair of high spin ferric and ferrous ions in the major reaction product of sulfide with the met form of the non-heme iron oxygen-carrying protein hemerythrin. Evidence for an analogous selenide derivative is also reported. Mössbauer and EPR spectroscopy establish (a) the charge and spin states of the individual iron atoms in sulfidehemerythrin as Fe(III), S = 5/2, and Fe(II), S = 2, and (b) the existence of an antiferromagnetic exchange interaction that couples the two spins to a resultant spin S = 1/2. The combined Mössbauer and EPR data confirm the correctness of the formulation first proposed for semi-methemerythrin by Harrington, P.C., de Waal, D.J.A., and Wilkins, R.G. ((1978) Arch. Biochem. Biophys. 191, 444-451) and furthermore show that a majority of the iron centers in the protein can be stabilized at this oxidation level. The results also demonstrate a new route to semi-methemerythrin. A titration of methemerythrin with selenide indicates that this derivative forms by a two step process consisting of first, reduction to the semi-met oxidation level by selenide and second, binding of selenide to either one or both irons.