Analysis of powder and single crystal electron paramagnetic resonance spectra of deoxy myoglobins containing cobalt (II) proto-and mesoporphyrins IX at various microwave frequencies

Artificial myoglobins (Mbs) substituted for protoheme with Co(II) proto-and mesoporphyrins IX (proto-and meso-CoMbs, respectively) were prepared. The principal values and eigenvectors of g tensors and the hyperfine coupling tensors of the paramagnetic Co(II) centers of their deoxy forms have been determined by single crystal EPR spectroscopy at 77 K in order to elucidate orientation and electronic structure of the prosthetic group in myoglobin. The orientation of the porphyrin plane of deoxy meso-CoMb were found to be identical to that of deoxy proto-CoMb. However, the in-plane hyperfine coupling constants of deoxy meso-CoMb were more anisotropic and larger than those of deoxy proto-CoMb, suggesting an increase in the electron spin density on the Co(II) ion upon the exchange of protoporphyrin IX with mesoprophyrin IX. Powder EPR spectra of these CoMbs, which were measured at S- and L-band microwave frequencies, exhibited well resolved 59Co hyperfine splittings and can be clearly interpreted by the use of the EPR parameters obtained from single crystal EPR measurements.     

1H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin.

The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by 1H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The 1H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated alpha-chain prior to assembling with the native beta-subunit to yield a tetramer with sulfhemin in the alpha-subunits. The subunit peak assignments establish that it is the beta-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.     

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.     

Replacement of the methionine axial ligand in cytochrome c550 by a lysine: effects on the haem electronic structure

The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His–Met cytochromes to His–Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c₅₅₀. The electronic structure of the His–Lys haem is shown to be similar to that produced by His–cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand.     

On the structure of the prosthetic group of citrate (pro-3S)-lyase.

The prosthetic group of citrate (pro-3S)-lyase from Klebsiella aerogenes as well as Streptococcus diacetilactis was obtained eigher by beta elimination or pronase digestion of the enzyme and purified by DEAE-cellulose chromatography. The compound was shown to contain 3 mol of PO4, 2 mol of ribose, and 1 mol of sulfhydryl/mol of adenine. 5'-AMP and dephospho-CoA are components of the prosthetic group. The evidence obtained so far support our proposed structure of 3' (or 2') leads to 1'-(5'-phosphoribosyl)dephospho-CoA for the prosthetic group of citrate lyase. The presence of one phosphomonoester group in the compound isolated after beta elimination and the absence of the same in the compound isolated after pronase digestion indicated that the prosthetic group is attached to the enzyme through a phosphodiester bond. Analyses of the pyruvate released by beta elimination and subsequent acid hydrolysis of the peptide-bound prosthetic group and its degradation products showed that the phosphodiester linkage is between the hydroxyl group of a serine residue of the protein and the 5'-PO4 group of the second ribose.     

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.     

A nuclear Overhauser effect study of the active site of myeloperoxidase. Structural similarity of the prosthetic group to that on lactoperoxidase

The low-spin, cyanide-ligated ferric complex of the intact bovine granulocyte myeloperoxidase (MPO-CN) has been studied by proton nuclear magnetic resonance utilizing the nuclear Overhauser effect (NOE). This is the largest globular protein (approximately 1.5 x 10(5) for the intact alpha 2 beta 2 tetrameric species) for which successful NOEs have been observed without serious interference of spin diffusion, and demonstrably confirms the utility of such studies on large paramagnetic as compared to diamagnetic proteins. The 1H NMR spectrum of MPO-CN is found to have a remarkable similarity in the number, resonance pattern, and metal ion-induced relaxation properties of the resolved, hyperfine-shifted resonances to those reported earlier for the analogous complex of bovine lactoperoxidase (LPO-CN); moreover, the interproton connectivities between pairs of hyperfine-shifted proton sets, as reflected by the NOEs, are also essentially the same (Thanabal, V., and La Mar, G. N. (1989) Biochemistry 28, 7038-7044). Since the extracted prosthetic group of lactoperoxidase is a porphyrin with proposed functionalization of the 8-methylene group (Nichol, A. W., Angel, L. A., Moon, T., and Clezy, P. S. (1987) Biochem. J. 247, 147-150), we interpret the resultant similarity in 1H NMR spectral parameters for LPO-CN and MPO-CN as indicating that the prosthetic groups in MPO and LPO are very similar, and hence likely both porphyrins with a similarly functionalized periphery that allows covalent linkage to the protein matrix. The hyperfine shift pattern of the broadest resolved lines lead to their assignment to the axial histidyl imidazole side chain. Two pairs of resonances are found to have similar relaxation properties and/or dipolar as similarly shifted resonances that arise from a distal His and Arg in horseradish peroxidase (as also found in LPO-CN), and suggest that MPO also possesses these catalytically functional residues in the distal heme pocket.     

Apparent hydrogen bonding by strongly immobilized spin-labels

The hyperfine separations of nitroxide spin-labels which are tightly bound within hemoglobin exhibit a substantial temperature dependence even when the hemoglobin is immobilized by freezing or precipitation. It is shown that NO.--HX hydrogen bond formation by the spin-label within its binding site is a good explanation for the observed temperature dependence. Comparative studies using different hemoglobin derivatives and two different spin-labels suggest that the HX group may be some element of the protein matrix and that this hydrogen bond may be a factor in the stabilization of the label within its binding site. The hyperfine separation of a fatty acid spin probe incorporated into aqueous bilayer dispersons of dipalmitoylphosphatidylcholine also exhibits a temperature dependence at low temperature which is qualitatively similar to that of the spin-labeled hemoglobin systems. Saturation transfer electron paramagnetic resonance measurements indicate that label motion is not the source of this temperature dependence. A hydrogen-bond equilibrium between water molecules and the nitroxide NO. group appears to be a plausible source of the temperature-dependent hyperfine separation in the lipid bilayer system. Small amplitude torsional oscillation or librational motion by the nitroxide may also produce additional changes in the hyperfine separation which are difficult to distinguish from hydrogen-bonding effects under some circumstances. The apparent hydrogen-bond equilibrium exhibits a strong thermal and environmental dependence which may be of importance in a number of biophysical spin-label measurements.     

Lactoperoxidase haem, an iron-porphyrin thiol.

The haem prosthetic group of lactoperoxidase can be prepared from the enzyme in high yield by reductive cleavage with mercaptoethanol in 8 M-urea under mild conditions. The product yields porphyrins, after removal of iron, which show visible spectroscopic properties similar to protoporphyrin but are considerably more polar. In the presence of iodoacetamide, a different product is obtained by reductive cleavage. The proton n.m.r. and mass spectra of this compound indicate that the prosthetic group of the enzyme is the iron complex of 18-mercaptomethyl-2,7,12-trimethyl-3,8-divinylporphyrin-13,17-d ipropionic acid. It is proposed that the unusual strength of binding of the prosthetic group to the apoprotein is due to formation of a disulphide bond from a cysteine residue to the porphyrin thiol.     

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.     

Phosphopantethenylated Precursor Acyl Carrier Protein Is Imported into Spinach (Spinacia oleracea) Chloroplasts

Acyl carrier protein (ACP) is an essential cofactor of fatty acid synthase. In plants, ACP is synthesized in the cytosol as a larger precursor protein and then is imported into the plastid where it is processed to a smaller mature form. The active form of ACP uses a covalently linked 4[prime]-phosphopantetheine prosthetic group derived from coenzyme A to covalently bind the acyl intermediates during fatty acid synthesis. The prosthetic group is added to ACP by holoACP synthase. This enzyme activity is associated with both the plastidial subcellular fraction and the soluble, or cytoplasmic, fraction. To gain further insight into potential in vivo pathways for the synthesis and maturation of ACP, in this study we examined whether precursor holoACP can be imported by isolated spinach (Spinacia oleracea) chloroplasts. Precursor holoACP containing a [35S]phosphopantetheine prosthetic group was prepared, and the radiolabel was used to demonstrate import of the phosphopantethenylated protein into isolated chloroplasts. In addition, timed chloroplast import assays indicated that in vitro import of the phosphopantethenylated protein is at least as efficient as import of the precursor apoprotein. Evidence was also obtained for a low level turnover of the prosthetic group among endogenous plastidial ACPs when coenzyme A was supplied exogenously.     

Acyl carrier protein from Escherichia coli: characterization by proton and fluorine-19 nuclear magnetic resonance and evidence for restricted mobility of the fatty acid chain in tetradecanoyl-acyl-carrier protein.

The acyl-carrier protein (ACP) of Escherichia coli is a protein of molecular weight 8847 with a 4'-phosphopanthetheine prosthetic group. ACP functions (via the SH of the prosthetic group) as a coenzyme in the synthesis of fatty acids and complex lipids. We report proton nuclear magnetic resonance (NMR) studies of the structure of ACP under various experimental conditions. The motion of the fatty acyl chain of acyl-ACP has been investigated by 19FNMR studies of difluorotetradecanoyl-ACP. 31PNMR studies of the prosthetic group phosphorus of ACP and acyl-ACP are also reported. We make the following conclusions: (1) the structure of ACP is stabilized by surface charge, and (2) the fatty acid residue of acyl-ACP does not move freely and seems immobilized by an interaction with the protein moiety.     

Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of α-tocopherol and its model compound in solution

The electron nuclear double resonance (ENDOR) spectra of chromanoxyl radicals obtained by the PbO₂ oxidation of α-tocopherol and its model compound were observed in t-butylbenzene, and the proton hyperfine coupling constants were correctly determined. Each of the two β- and γ-methylene protons in the chromanoxyl ring shows an equivalent hyperfine splitting, suggesting that the heterocyclic ring attached to the aromatic ring are coplanar with the plane of the aromatic system. A comparison of the hyperfine couplings in α-tocopheroxyl radical and its model shows that the introduction of a long-isoprenoid-chain in the α-tocopherol in place of a methyl group in the model compound has very little effect on the unpaired spin distribution or molecular structure of the chromanoxyl skeleton. The results of McLachlan molecular orbital (MO) calculations were found to be in satisfactory agreement with the ‘experimental’ spin densities evaluated from the hyperfine coupling constants.     

The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization

The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed.     

Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein

Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active. The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion. This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments. The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A. At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation. These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.     

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

The prosthetic group of citrate-lyase acyl-carrier protein.

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.     

Axial pertubations on the electronic and magnetic properties of ferric porphyrins: II. Solvent effects on the proton NMR spectra of low-spin cyano complexes

The proton NMR spectra of a series of low-spin bis-cyano ferric complexes of tetraarylporphyrins and octaethylporphyrin in a variety of solvents have been recorded and analyzed. The hyperfine shifts are shown to be very sensitive to the solvent, experiencing an overall downfield bias as the solvent hydroge-bonding donor strength increased. The characteristic pattern of the contact and dipolar shifts for the meso-aryl group in tetraarylporphyrin complexes are shown to permit a quantitative separation of the dipolar and contact contributions to the hyperfine shift. The separated components indicate that increased solvent hydrogen bonding strength significantly decreases the magnetic anisotropy of the iron and diminishes porphyrin → iron π bonding. The changes in anisotropy with solvent are shown to be consistent with the coordinated cyanide acting as a proton acceptor. Although similar effects are found to be absent in bis-imidazole complexes, a downfield bias of half the magnitude of the bis-cyano complexes is observed in mixed cyano/imidazole complexes. Hence, the heme hyperfine shifts in cyano-metmyoglobins and -hemoglobins may serve as probes for the protonation of the distal histidyl imidazole.     

Distance between lysine 384 and heme of cytochrome P-450 LM2 (P-450 IIB4) studied by fluorescence energy transfer measurements

The distance between FITC-modified lysine 384 of cytochrome P-450 LM2 and the active site, heme, was estimated by fluorescence energy transfer measurements. To avoid differential labelling of P-450 LM2 for protection of the alpha-amino group from FITC modification, deconvolution of measured fluorescence decay curves using a double exponential model was performed. A value of 2.7 nm was obtained for the distance FITC (lysine 384) - heme. This distance is too large to account for a direct electron tunneling from prosthetic group to prosthetic group at this interaction site between reductase and P-450 LM2.     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.