Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

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.     

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.     

Mixed-valence cytochrome oxidase-formate complex. A steady-state intermediate.

At neutral pH, formate binds to the haem a3 component of cytochrome c oxidase to give a complex that reacts differently from the non-liganded enzyme with reducing agents. Addition of sodium dithionite to the formate complex leads directly to the formation of the fully reduced species, whereas reduction with ascorbate/tetramethylenephenylene-diamine can lead to the production of a mixed-valence species. The stability of this mixed-valence form was studied, and the species appears to represent a 'steady-state' situation that is stable only in the presence of an excess of O2 and reducing equivalents. Characterization of the mixed-valence complex by electron paramagnetic resonance and magnetic circular dichroism reveals the presence of reduced low-spin haem a together with reduced detectable copper and high-spin ferric haem a3.     

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.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site.

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.     

Ligand-induced spectral changes in cytochrome c oxidase and their possible significance.

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.     

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.     

Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3

Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a3 centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm. Spectroscopic characterization of the steady-state species.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a1c1 is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c-550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a1c1 and aa3-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c-550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP+ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi. The electron transfer against redox potential from NO2- to cytochrome c could be pushed through prompt removal by cytochrome aa3 of H+ formed by the dehydrogenation of NO2- + H2O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO2-, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a1c1 with NO2- in vivo.     

Intramitochondrial positions of cytochrome haem groups determined by dipolar interactions with paramagnetic cations.

E.p.r.(electron-paramagnetic-resonance) spectra of the ferricytochromes were studied in normal and 'nickel-plated' pigeon heart mitochondria and pigeon heart submitochondrial particles. NiCL2 added to either mitochondria or particles was bound completely to the membranes, but none was transported across the vesicles. Hence, any perturbations of the haem e.p.r. spectra by Ni(II) should occur only for those cytochromes in close proximity to the exterior surface. Whenever Ni(II) can approach to within 1 nm of cytochrome haem. the consequent acceleration of the haem e.p.r. relaxation kinetics should elicit dipolar line broadening. Relaxation acceleration should also increase the incident power level required to saturate the haem e.p.r. signal. In pigeon heart mitochondria, at least three e.p.r. resonances, attributable in part to cytochromes c1, bK and br, are observed at gz=3.3 resonance. In these submitochondrial particles, the peak at gz=3.5 is missing, and the resonance at gz=3.6 resolves into two components, neither of which is sensitive to added Ni(ii). Addition of free haemin (ferric, a paramagnetic anion) to intact mitochondria elicits the same e.p.r. signal changes as does a preparation of submitochondrial particles. Saturation curves for cytochrome oxidase obtained for e.p.r. spectra of the high-spin form (g = 6) and the low-spin form (gz=3.1) also reveal no effect of Ni(II) on the haem e.p.r. relaxation in either mitochondria or inverted submitochondrial particles. Further, Ni(II) fails to alter the spectra or saturation properties of cytochrome c in either mitochondria or submitochondrial particles therefrom. Only with a 50-fold molar excess of Ni(II) can one accelerate the e.p.r. relaxation of cytochrome c in aqueous solution, although other more subtle types of magnetic interactions may occur between the cytochrome and either Ni(II) or ferricyanide. Addition of haemin to mitochondria likewise failed to alter the e.p.r. characteristics of either cytochrome c or cytochrome oxidase. The present observations strongly suggest that cytochromes bK, br and c1 reside on the exterior surface of the inner mitochondrial membrane. On the other hand, we find no positive evidence for the location of cytochrome c or cytochrome oxidase haem groups within 1 nm of either membrane surface. Because of possible shielding effects from the protein moieties, however, we cannot unequivocally assign the location of the haem groups to the membrane interior. The present results are not inconsistent with the observations of other investigators who used different techniques. However, it is clear that any model of energy coupling in mitochondrial oxidative phosphorylation must account for the positioning of all the b-c cytochrome haem groups on the outside.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Binding of ligands and spectral shifts in cytochrome c oxidase

1. On addition of reductant (ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa(3)), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415nm, 445nm and 605nm, which slowly gives rise to the final ;half-reduced' species in whose spectrum the 415nm peak has disappeared and a new absorption is seen at 430-435nm. 2. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the ;low-spin' cyanide form with a peak at 432nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally ;mixed-spin', undergoes no further changes. 3. The Soret-peak shift of oxidized cytochrome a(3) which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605nm to 603nm. A partial blue shift of the alpha-peak occurs in the half-reduced sulphide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulphides [a(2+)a(3) (3+)HSR compounds, where R=CH(3), C(2)H(5) or (CH(3))(2)CH]. 4. Analogous, albeit less readily decipherable, spectroscopic effects with the ligands imidazole and alkyl isocyanides suggest that on reduction of cytochrome a an interaction occurs between the two haem groups involving (i) a high- to low-spin change in cytochrome a(3), and after this, (ii) a change in the molecular environment of the cytochrome a. The latter effect, possibly a decrease in the hydrophobicity of the haem pocket, requires that the ligands on cytochrome a(3) have a bulky and partially hydrophobic character.     

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.     

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.     

The absorbance coefficient of beef heart cytochrome c1

Isolated cytochrome c1 contains endogenous reducing equivalents. They can be removed by treating the protein with sodium dithionite followed by chromatography. This treatment has no effect on the reaction with cytochrome c, nor does it alter the optical spectrum, or the polypeptide or amino acid composition of the protein. Both the titration of dithionite-treated ferrocytochrome c1 with potassium ferricyanide and the anaerobic titration of dithionite-treated ferricytochrome c1 with NADH in the presence of phenazine methosulphate lead to the same value for the absorbance coefficient of cytochrome c1: 19.2 mM-1 . cm-1 at 552.4 nm for the reduced-minus-oxidised form. This value was also obtained when the haem content was determined by comparing the spectra of the reduced pyridine haemochromes of cytochrome c and cytochrome c1. Comparison of the optical spectra of cytochrome c and cytochrome c1 by integration shows equal transition moments for the transitions in the porphyrin systems of both proteins. A set of equations with which the concentration of the cytochromes aa3, b, c and c1 can be calculated from one reduced-minus-oxidised difference spectrum of a mixture of these proteins.