Assignment of the axial ligands of the haem in milk lactoperoxidase using magnetic circular dichroism spectroscopy.

The absorption and MCD spectra of ferric lactoperoxidase from milk and its cyanide and fluoride derivatives have been measured in the near infrared and visible wavelength regions both at room temperature and at 4.2 K. By comparison with the MCD spectra of haemoproteins of known axial ligation, which also contain protohaem IX, it has been possible to arrive at suggestions for the axial ligation in lactoperoxidase. At room temperature oxidized lactoperoxidase has the haem iron in the high-spin state, and the results indicate that the proximal ligand of the haem iron is a histidine imidazole and that the sixth ligand is probably a carboxylate ion. At 4.2 K oxidized lactoperoxidase converts almost totally to a low-spin form, changing the sixth ligand to a histidine imidazole, which is in the imidazolate form.

A comparative study of the low-temperature magnetic circular dichroism spectra of horse heart metmyoglobin and bovine liver catalase derivatives.

The magnetic circular dichroism (MCD) spectra of three horse heart metmyoglobin compounds, the cyanide, azide and hydroxide forms, have been measured in the visible and near infrared spectral regions at temperatures down to 1.5 K. The three compounds are all virtually completely low-spin at low temperatures with ground g factors of decreasing rhombicity in the order CN- greater than N3- greater than OH-. The MCD magnetization curves have been constructed at selected wavelengths throughout the visible and near infrared regions. The curves are independent of wavelength, showing that all the bands studied are x,y polarized and can, moreover, be satisfactorily fitted to the g factors determined by EPR spectroscopy with theoretical expressions (Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191, 411-420). This confirms the assignment and polarizations of the near infrared region low-spin ferric haem charge-transfer bands. The energies of these transitions are markedly dependent upon the added axial ligand, ranging from 1595 to 1295, and 1050 nm for the compounds CN-, N3- and OH-. The MCD spectra of bovine liver catalase and its cyanide adduct have been recorded in the Soret, visible and near infrared regions. Catalase is know to have phenolate anion as the proximal ligand of the haem group. The forms of the spectra make an interesting comparison with those of the analogous metmyoglobin derivatives, in which histidine is the proximal ligand. The MCD spectra of catalase at 4.2 K is an example of a fully high-spin haemoprotein. The cyanide compound is completely low-spin at 4.2 K. The near infrared charge-transfer band is at 1300 nm, showing the effect on the energy of this band of changing from imidazole to phenolate ion as the proximal ligand to haem.

Redox properties of the diheme cytochrome c4 from Azotobacter vinelandii and characterisation of the two hemes by NMR, MCD and EPR spectroscopy.

From biphasic stopped-flow kinetic studies it has been established that the two heme centres of cytochrome c4 from Azotobacter vinelandii undergo redox change with [Co(terpy)2]3+/2+ (260 mV) at different rates. Rate constants for oxidation and reduction at pH 7.5 give reduction potentials for the two heme centres in agreement with previous values from spectrophotometric titrations (263 and 317 mV). From NMR studies on the fully reduced protein two sharp methyl methionine resonances are observed at -3.16 and -3.60 ppm, consistent with axial methionine coordination. On titration with [Fe(CN)6]3- the -3.16 ppm resonance is the first to disappear, and is assigned to the less positive reduction potential. Line-broadening effects are observed on partial oxidation, which are dominated by intermolecular processes in an intermediate time-range exchange process. The hemes of the oxidised protein are distinguishable by EPR g-values of 3.64 and 3.22. The former is of interest because it is at an unusually low field for histidine/methionine coordination, and has an asymmetric or ramp shape. The latter assigned to the low potential heme is similar to that of a cytochrome c551. The MCD spectra of the fully oxidised protein are typical of low-spin Fe(III) heme centres, with a negative peak at 710 nm characteristic of methionine coordination, and an NIR peak at 1900 nm characteristic of histidine/methionine (axial) coordination. Of the four histidines per molecule only two undergo diethyl pyrocarbonate (DEPC) modification.

Spectroscopic studies of partially reduced forms of Wolinella succinogenes nitrite reductase.

Reductive titrations of the dissimilatory hexa-haem nitrite reductase, Wolinella succinogenes, with methyl viologen semiquinone (MV) and sodium dithionite, have been followed at room temperature by absorption, natural (CD) and magnetic circular dichroism (MCD) spectroscopies and at liquid helium temperature by electron paramagnetic resonance (EPR) and MCD spectroscopies. The nature of the reduced enzyme depends on the reductant employed. At room temperature a single high-spin ferrous haem, observed by MCD after reduction with MV, is absent from dithionite reduced samples. It is suggested that a product of dithionite oxidation becomes bound with high affinity to the reduced state of the enzyme causing the ferrous haem to become low-spin. The site occupied is likely to be the substrate binding haem. The course of the titration with MV at room temperature shows the reduction of high-spin ferric to high-spin ferrous haem. Since the EPR spectrum reveals the presence of an unusual high-low spin ferric haem pair in the oxidised state we propose that the active site of the enzyme is a novel haem pair consisting of one high (5-coordinate) and one low-spin (6 coordinate) haem, magnetically coupled and possibly bridged by a histidinate ligand.

A comparison by magnetic circular dichroism of compound X and compound II of horseradish peroxidase.

The chlorite product of horseradish peroxidase, compound X, is shown by magnetic circular dichroism (MCD) spectroscopy in the temperature range 1.6-50 K to have a very similar haem structure to compound II under the same conditions (pH 10.7). Both are concluded to contain the Fe(IV) = 0 group. The MCD spectrum also detects an unusual species, absorbing at wavelengths between 600 and 750 nm, that has magnetic properties different from those of the ferryl haem group. It is suggested that this is a species at the same oxidation level as ferryl haem but with the porphyrin ring having suffered a one-electron oxidation, i.e. [Fe(III) P.+].

Electron paramagnetic resonance and magnetic circular dichroism studies of a hexa-heme nitrite reductase from Wolinella succinogenes.

The nature of the heme centers in the hexa-heme dissimilatory nitrite reductase from the bacterium Wolinella succinogenes has been investigated with EPR and magnetic circular dichroism spectroscopy. The EPR spectrum of the ferric enzyme is complex showing, in addition to magnetically isolated low-spin ferric hemes with g values of 2.93, 2.3 and 1.48, two sets of signals at g = 10.3, 3.7 and 4.8, 3.21, which we assign to two pairs of exchange coupled hemes. The MCD spectra show that the isolated hemes are bis-histidine coordinated and that there is one high-spin ferric heme. The exchange coupling is lost on treatment with SDS.

The structure of the cytochrome a3-CuB site of mammalian cytochrome c oxidase as probed by MCD and EPR spectroscopy.

The nature of the complexes formed between cytochrome c oxidase and the three inhibitory ligands N3-, CN-, and S2- have been investigated by a combination of MCD and EPR spectroscopy. CN- forms a linear bridge between the Fe III a3 and CuB II, suggesting that the distance between these centers in the oxidized enzyme is between 5 and 5.25 A. This distance is too short to permit N3- to form a linear bridge and the evidence suggests this to be bent. In contrast S2- or SH- is unable to form any bridge and it seems likely that two SH- ions are bound by the bimetallic site, one to Fe III a3 and the other to CuB I. The significance of the a3-CuB distance in terms of oxygen binding and reduction is discussed.

A study of the oxidized form of Pseudomonas aeruginosa cytochrome c-551 peroxidase with the use of magnetic circular dichroism.

The magnetic properties at different temperatures of oxidized Pseudomonas aeruginosa cytochrome c-551 peroxidase were studied, with the use of the technique of magnetic-circular-dichroism spectroscopy. At 4.2K, both constituent haems were found to be low-spin, and the axial ligand pairs were identified as histidine-histidine and histidine-methionine. At room temperature high-spin signals were observed, amounting to less than 25% of the total haem present. These signals are concluded to arise mainly from a temperature-dependent spin-state equilibrium in the methionine-ligated haem.

Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa.

Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.

pH-dependent forms of the ferryl haem in myoglobin peroxide analysed by variable-temperature magnetic circular dichroism.

The reaction product of myoglobin and H2O2 exists in two different forms according to the external pH. Varied-temperature magnetic-circular dichroism (m.c.d.) spectroscopy demonstrates that both contain the oxyferryl ion Fe(IV) = O. Alkaline myoglobin peroxide has often been used as a model for oxidized intermediates in the catalytic cycles of haem-containing peroxidases, but absorption and m.c.d. spectra show that the acid form is much more closely related to species such as horeradish peroxidase Compound II. The differences are tentatively ascribed to ionization of the proximal histidine ligand in alkaline myoglobin peroxide. It is also shown that the m.c.d. method allows an estimate of the zero-field splitting parameter of both forms, values of D = 28.0 +/- 3 cm-1 and 35.0 +/- 5 cm-1 being obtained for the alkaline and acid forms respectively.

The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes.

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.

The formation of ferric haem during low-temperature photolysis of horseradish peroxidase Compound I.

Illumination at low temperature of the peroxide compound of horseradish peroxidase (HRP-I) causes partial conversion of the haem electronic structure from a ferryl-porphyrin radical species into a low-spin ferric state. Magnetic-c.d. (m.c.d.) and e.p.r. spectral features of the photolysis product are almost identical with those of the alkaline form of ferric HRP, proposed on the basis of its near-i.r. m.c.d. spectrum to be a Fe(III)-OH species. The ferric product of HRP-I photolysis also contains free-radical e.p.r. signals. Conversion of HRP-I into the Fe(III)-OH species, which requires transfer of a proton and two electrons from the protein, is shown to be a two-step process.

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre.

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.

Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa.

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Characterization of haem and iron-sulphur centres by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy.

The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.

N.m.r., e.p.r. and magnetic-c.d. studies of cytochrome f. Identity of the haem axial ligands.

N.m.r.-, magnetic-c.d.- and e.p.r.-spectroscopic studies of oxidized and reduced cytochrome f from charlock, rape and woad are reported. Comparison of the spectra with corresponding spectra of other haem proteins, including horse and yeast cytochromes c, bovine cytochrome b5 and n-butylamine adduct of soya-bean leghaemoglobin support the hypothesis [Siedow, Vickery & Palmer (1980) Arch. Biochem. Biophys. 203, 101-107] that lysine is the sixth ligand of native cytochrome f. Detailed analysis of the e.p.r. spectrum of ferricytochrome f indicates that its principle g-values are 3.51, 1.70 and less than 1.3, and not 3.48, 2.07 and 1.6 as previously suggested [Siedow, Vickery & Palmer (1980) Arch. Biochem. Biophys. 203, 101-107]. The observation of a one-proton intensity resonance at -3.27 p.p.m. in the 1H-n.m.r. spectrum of ferrocytochrome f, coupled with the absence of a methionine methyl resonance from the spectral region to low frequency of -2 p.p.m., is suggested to be a general indicator of lysine co-ordination.

Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase.

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.