Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase.

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.     

Variable-temperature magnetic-circular-dichroism spectra of cytochrome c oxidase and its derivatives

A detailed study of the effect of temperature on the m.c.d. (magnetic circular dichroism) spectra of cytochrome c oxidase and some of its derivatives was undertaken to characterize the spin states of haem a and a(3). The fully reduced enzyme contains haem a(3) (2+) in its high-spin form and haem a(2+) in the low-spin state. This conclusion is reached by comparing the spectrum with that of the mixed-valence CO derivatives and its photolysis product. The cyanide derivative of the fully reduced enzyme contains both haem a and a(3) in the low-spin ferrous form. The m.c.d. spectra of the fully oxidized derivatives are consistent with the presence of one low-spin ferric haem group, assigned to a, which remains unaltered in the presence of ligands. Haem a(3) is high spin in the resting enzyme and the fluoride derivatives, and low spin in the cyanide form. The partially reduced formate and cyanide derivatives have temperature-dependent m.c.d. spectra due to the presence of high- and low-spin haem a(3) (3+) respectively. Haem a is low-spin ferrous in both. A comparison of the magnitude of the temperature-dependence of haem a(3) (3+) in the fully oxidized and partially reduced forms shows a marked difference which is tentatively ascribed to the presence of anti-ferromagnetic coupling in the fully oxidized form of the enzyme, and to its absence from the partially reduced derivatives, owing to the reduction of both Cu(2+) ions.     

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.     

A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.     

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 the assimilatory nitrite reductase from barley Hordeum vulgare leaves.

The magnetic-circular-dichroism (m.c.d.) spectra of oxidized 'resting' bovine cytochrome c oxidase and the cyanide-inhibited form are reported at 5.15 T and at 4.2 K along with m.c.d. magnetization curves plotted at selected wavelengths. In both spectra there are features at 790nm and 1564nm due to Cua and haem a respectively, the e.p.r.-detectable components of the enzyme. There is a new peak at 1946nm only in the spectrum of the cyanide-inhibited enzyme. Arguments are advanced that assign this to low-spin ferric haem a3 bridged to Cua3, thereby forming a ferromagnetically coupled pair of metal ions.     

Spectroscopic identification of the haem ligands of cellobiose oxidase.

A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra-red MCD techniques, has identified the axial ligands of the b-type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.     

Some magnetic properties of Pseudomonas cytochrome oxidase.

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.     

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.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.     

Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.     

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.     

The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy.

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.     

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.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

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.     

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.     

The structure of the dihaem cytochrome b of fumarate reductase in Wolinella succinogenes: circular dichroism and sequence analysis studies

The fumarate reductase from Wolinella succinogenes contains two haem groups with markedly different midpoint potentials (-20 mV and -200 mV). The enzyme is made up of three subunits, the lipophilic one of which (cytochrome b) ligates the haems. Circular dichroism (CD) spectroscopy has been applied to the reductase in order to obtain information on the structure of the haems and of their environment. This approach is integrated with amino acid sequence comparison of the cytochrome b with other quinone-reacting membrane haemoproteins for predicting the axial ligands of the haems as well as their location relative to the membrane. The following results have been obtained: (1) the CD spectra in the Soret region show exciton coupling indicating haem-haem interaction, which is particularly evident in the reduced state and disappears upon denaturation of the enzyme; (2) The apoprotein of cytochrome b is predicted to consist of five hydrophobic helices (helices A-D and cd), four of which should span the membrane. Helices A, B, C and cd contain a histidine residue each which possibly forms one of the ligands of the haems. It is proposed that haem b (-20 mV) is ligated by H44 and H93, and haem b (-200 mV) by H143 and H182.