Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Sch?fer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.     

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans

Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561-575 nm, 552-540 nm, and 602-630 nm indicated the presence of three b-type cytochromes (Em,7 of +244 +/- 8, +24 +/- 3, -163 +/- 11 mV), four c-type cytochromes (Em,7 of +280 +/- 10, +210 +/- 5, +125 +/- 8, and 20 +/- 3 mV) and two a-type cytochromes (Em,7 of +335 +/- 15, +218 +/- 18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50 = 2 microM) and azide (I50 = 1 mM), while a soluble cytochrome c (c551, Em,7 = +217 +/- 2 mV) was shown to be the physiological electron carrier connecting the bc1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh > or = +80 < or = +140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.     

EPR studies of the cytochrome-d complex of Escherichia coli

We have examined the thermodynamic and EPR properties of one of the ubiquinol oxidase systems (the cytochrome d complex) of Escherichia coli, and have assigned the EPR-detectable signals to the optically identified cytochromes. The axial high spin g = 6.0 signal has been assigned to cytochrome d based on the physicochemical properties of this signal and those of the optically defined cytochrome d. A rhombic low spin species at gx,y,z = 1.85, 2.3, 2.5 exhibited similar properties but was present at only one-fifth the concentration of the axial high spin species. Both species have an Em7 of 260 mV and follow a -60 mV/pH unit dependence from pH 6 to 10. The rhombic high spin signal with gy,z = 5.5 and 6.3 has been assigned to cytochrome b-595. This component has an Em7 of 136 mV and follows a -30 mV/pH unit dependence from pH 6 to 10. Lastly, the low spin gz = 3.3 signal which titrates with an Em7 of 195 mV and follows a -40 mV/pH unit dependence from pH 6 to 10 has been assigned to cytochrome b-558. Spin quantitation of the high-spin signals indicates that cytochrome d and b-595 are present in approximately equal amounts. These observations are discussed in terms of the stoichiometry of the prosthetic groups and its implications on the mechanism of electron transport.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR.

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em = +280 mV, 30 degrees; gz = 3.33, Em = 0 mV, 45 degrees; gz = 3.03, Em = +95 mV, 40-50 degrees and gz = 2.95, Em = +150 mV, 90 degrees. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the +95 mV and the +150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.     

The electronic state of heme in cytochrome oxidase II. Oxidation-reduction potential interactions and heme iron spin state behavior observed in reductive titrations.

Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and optical absorption spectroscopies have been used to monitor the concentrations of oxidized and reduced heme and copper during stoichiometric reductive titrations of purified beef heart cytochrome oxidase. The MCD data are deconvoluted to obtain the concentrations of reduced cytochromes a and a3 during the titrations; analysis of the EPR spectra provides complementary data on the concentrations of the EPR-detectable species. For the native enzyme in the absence of exogenous ligands, cytochromes a and a3 are reduced to approximately the same extent at all points in the titration. The reduction of the EPR-detectable copper, on the other hand, initially lags the reduction of the two cytochromes but in the final stages of the titration is completely reduced prior to either cytochrome a or a3. These non-Nernstian titration results are interpreted to indicate that the primary mode of heme-heme interaction in cytochrome oxidase involves shifts in oxidation-reduction potential for each of the two cytochromes such that a change in oxidation state for one of the hemes lowers the oxidation-reduction potential of the second heme by approximately 135 mV. In these titrations high spin species are detected which account for 0.25 spin/oxidase maximally. Evidence is presented to indicate that at least some of these signals can be attributed to cytochrome a3+ which has undergone a low-spin to high-spin state transition in the course of the titration. In the presence of carbon monoxide the oxidation-reduction properties of cytochromes a and a3 are markedly altered. The a32+. CO complex is fully formed prior to reduction of either cytochrome a3+ or the EPR-detectable copper. The g = 3 EPR signal attributed to cytochrome a3+ decreases as the MCD intensity of cytochrome a2+ increases; no significant high-spin intensity is observed at any intermediate stage of reduction. We interpret these Nernstian titration results to indicate that in the presence of ligands the oxidation-reduction potential of cytochrome a relative to cytochrome a3 is determined by the oxidation-reduction state of the stabilized cytochrome a3 ligand complex; if ligand binding occurs to reduced cytochrome a3 then cytochrome a titrates with a lower potential; cytochrome a titrates with a higher potential if oxidized cytochrome a3 is stabilized by ligand binding.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

Properties of the two terminal oxidases of Escherichia coli.

Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.     

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ

The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy.

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g approximately 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undectable components reduced appears to be inverely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during yhe titration. At pH 9.3 the quantity of heme represented in the high-spin signals is less than 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex.

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a1, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minus-oxidized split-alpha band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a1 (Em = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the Em of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.     

A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon

Abstract Membranes of the extremely thermoacidophilic archaeon Desulfurolobus ambivalens grown under aerobic conditions contain a quinol oxidase of the cytochrome aa ₃-type as the most prominent hemoprotein. The partially purified enzyme consists of three polypeptide subunits with apparent molecular masses of 40, 27 and 20 kDa and contains two heme A molecules and one copper atom. CO difference spectra suggest one heme to be a heme a ₃-centre. The EPR spectra indicate the presence of a low-spin and a high-spin heme species. Redox titrations of the solubilized enzyme show the presence of two reduction processes, with apparent potentials of + 235 and + 330 mV. The enzyme cannot oxidize reduced cytochrome c , but rather serves as an oxidase of caldariella quinone. Due to their very simple composition, D . ambivalens cell appear as a promising candidate to study Structure-function relationships of cytochrome aa ₃ in the integral membrane state.     

Biochemical and biophysical properties of cytochrome o of Azotobacter vinelandii

Cytochrome o, solubilized from the membrane of Azotobacter vinelandii, has been purified to homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The detergent-containing cytochrome o is composed of one polypeptide chain with a molecular weight of 28 000-29 000, associated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exists as a dimer by gel filtration analysis. The amino analysis which reveals the majority of residues are of hydrophobic nature. The cytochrome o oxidase contains protoheme as its prosthetic group and about 20-40% of phospholipids. The phospholipids are identified as phosphatidylethanolamine and phosphatidylglycerol by radioautographic analysis using 2-dimensional thin-layer chromatography. No copper or nonheme iron can be detected in the purified oxidase preparation by atomic absorption and chemical analyses. Oxidation-reduction titration shows this membrane-bound cytochrome o to be a low-potential component, and Em was determined to be -18 mV in the purified form and -30 mV in the membrane-bound form. Both forms bind CO with a reduced absorption peak at 559 and 557-558 nm in the native and solubilized forms, respectively. A high-spin (g = 6.0) form is assigned to the oxidized cytochrome o by electron paramagnetic resonance analysis, and KCN abolishes this high-spin signal. CO titration of purified cytochrome o in the anaerobic conditions shows the enzyme binds one CO per four protohemes and a dissociation constant is estimated to be 3.2 microM for CO. Cyanide reacts with purified cytochrome o in both oxidized and CO-bound forms, identified by specific spectral compounds absorbed at the Soret region. Cytochrome c, often co-purified with cytochrome c from the membrane, cannot serve as a reductant for cytochrome o in vitro, due to the apparent potential difference of about 300 mV. Upon separation, both cytochrome o and cytochrome c4 show a great tendency of aggregation. Furthermore, the oxidase activity (measured by tetramethyl-p-phenylenediamine oxidation rate) decreases as the cytochrome c concentration is decreased by ammonium sulfate fractionation. All these suggest the structural and functional complex nature of cytochrome c4 and cytochrome o in the membrane of A. vinelandii.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.