Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

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.     

Prosthetic group content and ligand binding properties of a spinach catalase

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.     

The prosthetic group of methylamine dehydrogenase from Pseudomonas AM1: evidence for a quinone structure

The g-value and linewidth of ESR spectra of methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating) EC 1.4.99.-) and methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) are very similar. This similarity is also reflected in electron-nuclear double resonance (ENDOR) results, the coupling constants of two protons in one enzyme equalling those in the other. The presence of a third proton in the ENDOR spectrum of methylamine dehydrogenase suggests a different structure or a different kind of interaction which can be related to the finding that the resolved ROSTHETIC GROUP IS PROTEIN-BOUND. The bound prosthetic group has a high redox-potential, supporting the conclusion from the ESR and ENDOR results that it is a quinone derivative.     

Selenium binding to beef-kidney rhodanese.

The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium-treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found. The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometric selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione. By comparison with model complexes the selenium-rhodanese intermediate was identified as a cysteinyl-selenium derivative.     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Quantitative similarities in the several actions of cyanide on prostaglandin H synthase

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a KI value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflected an increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a KI of 4 mM. The higher KI value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Oxidation-reduction properties of maize ferredoxin: sulfite oxidoreductase

Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced siroheme group of the enzyme. The effects of siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.     

One- and two-dimensional 1H NMR study of the substance P fragment ARG-PRO-Lys-Pro

The Substance P fragment Arg1-Pro2-Lys3-Pro4 (SP1-4) has been extensively investigated by means of proton nuclear magnetic resonance at 400 MHz. The combined application of different 2D techniques and a comparison of SP1-4 with its derivative SP1-4-amide allowed the complete and unambiguous assignment of the proton NMR spectrum. Conformational data obtained from the different NMR parameters are compared with theoretical calculations. The results suggest that SP1-4 exists, at the chosen experimental conditions, as a stretched molecule.     

The VI international conference on prostaglandins and related compounds

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a K_I value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflect and increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a K_I of 4 mM. The higher K_I value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Purification and partial characterization of microsomal cytochrome b555 from the higher plant Catharanthus roseus

Microsomal b-type hemoprotein designated, cytochrome b555 of C.roseus seedlings was solubilized using detergents and purified by a combination of ion exchange chromatography and gel filtration to a specific content of 18.5 nmol per mg of protein. The purified cytochrome b555 was homogeneous and estimated to have an apparent molecular weight of 16500 on SDS-PAGE. The absorption spectrum of the reduced form has major peaks at 424, 525 and 555 nm. The alpha-band of the reduced form is asymmetric with a pronounced shoulder at 559 nm. The spectrum of the pyridine ferrohemochrome shows absorption peaks at 557, 524 and 418 nm indicating that the cytochrome has protoheme prosthetic group. The purified cytochrome is autoxidizable and does not combine with carbon monoxide, azide or cyanide. It is reducible by NADH in the presence of NADH-cytochrome b555 reductase partially purified from C.roseus microsomes.     

Reaction of formate with the fast form of cytochrome oxidase: a model for the fast to slow conversion.

The ability to isolate preparations of cytochrome oxidase which are highly homogeneous has facilitated a study of the effects of various reagents on the purified enzyme. The addition of either sodium formate, formamide, formaldehyde, or sodium nitrite to enzyme which reacts in a single rapid kinetic phase with cyanide causes a blue-shift of 4-6 nm of the net (cytochrome a + cytochrome a3) Soret maximum. Only the derivative prepared by adding sodium formate demonstrates measurable intensity in the g' = 12 region of the low-temperature electron paramagnetic resonance (EPR) spectrum. This g' = 12 resonance is characteristic of cytochrome oxidase which has undergone a modification at the binuclear center and thereby reacts sluggishly with cyanide. As the site of cyanide binding in resting enzyme as been demonstrated to be CuB [Yoshikawa, S., & Caughey, W.S. (1990) J. Biol. Chem. 265, 7945-7958], it is proposed that formate can bind to CuB and the fast to slow transition is rationalized by using this proposal. The g' = 12 signal is also produced upon the addition of sodium formate to mitochondrial preparations, suggesting that the species responsible for this behavior may have possible physiological relevance. Physical properties of the formate derivative and data for other reagents reacted with the fast-reacting enzyme preparation are presented.     

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.     

Labile sulfur in human Superoxide dismutase.

1. The nautre of the intense absorption band at 320 nm of the copper and zinc-containing enzyme superoxide dismutase, from human red blood cells, has been investigated. The band does not depend on the metal prosthetic groups of the enzyme, as it is still present in the apo protein. When, however, copper alone is removed from the enzyme with a treatment involving the use of cyanide, the band is also lost. Nevertheless the copper-free protein is able to recover both the enzyme activity and the native electron paramagnetic resonance spectrum as easily as the apo protein. 2. A number of other treatments are able to abolish the band. They include reaction with reducing agents such as dithiothreitol, sulfite, borohydride, exposure to denaturants such as guanidine HCl and sodium dodecyl sulfate, and exposure to pH values below pH 3 or above pH 13. 3. Four sulfur atoms per protein molecule were found to be associated to the 320-nm chromophore on the basis of quantitative determinations following reaction with cyanide or sodium borohydride. 4. A molar absorption coefficient of 1150 M-1 cm-1 was determined per each chromophoric group. In spite of this relatively high value and unusual stability, a persulfide group, R-S-SH, seems to be the most likely structure for this chromophore. 5. Bovine and equine superoxide dismutase do not show spectral or chemical evidence for such a group. This, and the recovery of activity and spectral properties of copper in the cyanide-treated human enzyme, indicate that labile sulfur is not associated with the superoxide dismutase activity of this protein.     

ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein.

Electron nuclear double resonance (ENDOR) signals have been obtained from iron-linked nitrogens in frozen solutions of cytochrome c, metmyoglobin cyanide, and a low spin protohemin mercaptide complex. Hyperfine couplings from heme protons have also been obtained from metmyoglobin cyanide and from a low spin protohemin cyanide complex. Several of these proton resonances are assigned to specific heme protons.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

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.     

Structure of methylmenaquinone-7 isolated fromAlteromonas putrefaciens IAM 12079

The structure of methylmenaquinone-7, a major component of methylmenaquinones isolated fromAlteromonas putrefaciens IAM 12079, was determined by comparing the proton nuclear magnetic resonance (¹H-NMR) spectrum of its chromenyl derivative with those of menachromenyl acetate prepared from menaquinone-7 and phyllochromenyl acetate. Based on the numbering system used for naphthopyrane nucleus, proton signals of the 7- and 10-position for chromenyl derivatives were assigned from the signals on the¹H-NMR spectra phyllochromenyl acetate and phyllochromenol. As a result, the chemical structure of methylmenaquinone-7 was determined as 2,8-dimethyl-3-farnesylgeranygeranyl-1,4-naphthoquinone.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.