Purification and reconstitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis.

The hydroxylation of 6-deoxyerythronolide B (6D) to erythronolide B, a step in the biosynthesis of the 14-membered macrolide antibiotic erythromycin A by Saccharopolyspora erythraea, is catalyzed by a cytochrome P-450 monooxygenase that requires two electron transport proteins for the function of this terminal hydroxylase (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210, 1987). Two flavoproteins and an iron-sulfur protein (erythrodoxin) were purified from S. erythraea CA340 and shown to act with 6D hydroxylase to catalyze the hydroxylation of (9R)-[9-3H]9-deoxo-9-hydroxy-6D in vitro in a suitably reconstituted system. The flavoproteins contained flavin adenine dinucleotide and exhibited characteristic absorption maxima at 356 and 456 nm. The one with an Mr of 47,000 showed NADPH-dependent diaphorase and cytochrome c reductase activity, and the other, with an Mr of 53,000 showed NADH-dependent activities of the same two types. Erythrodoxin contained acid-labile sulfur and iron, had an Mr of 27,500, and showed a broad absorption maximum between 394 and 404 nm. The sequence of its first 15 amino acids, except for position 12, was the same as that of the ferredoxin from Mycobacterium smegmatis.     

The electron transport system of the anaerobic Propionibacterium shermanii: cytochrome and inhibitor studies.

1. Electron transport particles obtained from cell-free extracts of Propionibacterium shermanii by centrifugation at 105000 times g for 3 hrs oxidized NADH, D,L-lactate, L-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too. 2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome alpha1 and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80--90%. 3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or "site I region", in the electron transport system of P. shermanii. 4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor and reached 80--100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase. 5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions. 6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.     

Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium.

Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.     

Characterization of cytochrome b5 reconstituted with a ferric chlorin and a ferric oxochlorin

The role of the electronic properties of the heme group of rat cytochrome b5 in biological electron transfer was investigated by substituting chlorin analogues for the native protoporphyrin IX prosthetic group. The resultant purified proteins displayed physical and chemical properties distinct from those of the native enzyme. Optical spectroscopy of the ferric chlorin substituted cytochrome b5 revealed a blue-shifted Soret at 404 nm and a band at 586 nm characteristically red-shifted from the protohemin absorption band. The reduced, reconstituted protein displayed maxima at 406, 418, 563, and 600 nm. The oxidized cytochrome b5 containing the oxochlorin analogue produced a red-shifted Soret with maxima at 338, 416, and 602 nm. The reduced species differed only in the visible region with absorption maxima at 508, 554, and 600 nm. Characterization by EPR spectroscopy of the oxochlorin-substituted cytochrome b5 yielded g values of 2.566, 2.375, and 1.756 and respective axial delta/lambda and rhombic V/lambda components of 2.857 and 3.287, indicating significant electronic distortion in the chlorin ring and an increase in electron donation from the axial histidine ligands. A decrease in the reduction potential of 52 +/- 5 mV (50 mM KPi, pH 7.0, 25 degrees C) for the chlorin-reconstituted cytochrome b5 was determined with respect to that of native cytochrome b5. The reduction potential for the oxochlorin-containing cytochrome b5 was unchanged from that of the native system. Both of the reconstituted proteins were found to be capable of transferring electrons to cytochrome c in a reconstituted system dependent on NADH and cytochrome b5 reductase, thus stimulating the activity of native cytochrome b5.     

Purification and properties of cytochrome b-562.5 from Ulva pertusa.

Cytochrome b-562.5 (Ulva pertusa) was extracted from a green alga, U. pertusa, by homogenization of the thalli in phosphate buffer solution. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, and Sephadex gel filtration. Cytochrome b-562.5 has absorption maxima at 562.5 (alpha), 530.5 (beta), 429 (gamma), and 326 nm (delta) in the reduced form and at 537, 415 (gamma), and 275 nm in the oxidized form. The alpha-band of the reduced form is asymmetric with a shoulder at 560 nm, at liquid nitrogen temperature this band splits into two distinct peaks at 562 and 556.5 nm. The absorption maxima of the pyridine ferrohemochrome appear at 556 (alpha), 523 (beta), and 418 nm (gamma). The cytochrome does not combine with carbon monoxide or cyanide. The preparation of the cytochrome shows little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.20 V at pH7.0. The molecular weight of the cytochrome was estimated by Sephadex gel filtration to be 23x10(3).     

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.     

Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552.5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135,000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged from +234 mV to +242 mV, except for the heme d1 of component I (Em7 = +134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c-551 and c-552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c-552 to component II (Km = 1.27 microM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. III. Light-Induced Absorbance Changes in Chloroplast Fragments of the Wild Type and Mutant Strains

Light-induced absorbance changes were investigated in chloroplast fragments of wild type Chlamydomonas reinhardi and 5 different mutant strains having impaired photosynthesis. Two absorbance changes were detected, 1 having a maximum at 553 nm and the other at 559 nm. The component exhibiting the 553 nm change is a cytochrome similar to cytochrome f from higher plant chloroplasts. The component exhibiting the 559 nm change has the properties of a cytochrome similar to cytochrome b(3). Two of the mutant strains (ac-115 and ac-141) were found to lack the 559 cytochrome and light induced only the oxidation of the 553 cytochrome. A third mutant strain (ac-206), previously shown to lack the 553 cytochrome, exhibited only the light-induced reduction of the 559 cytochrome. A fourth mutant strain (ac-208), shown to lack plastocyanin, exhibited absorbance changes attributable to both cytochromes. However, light was capable of inducing the reduction of the 559 cytochrome but not its oxidation. On the other hand, light induced the oxidation of the 553 cytochrome but not its reduction.These observations are discussed in terms of the series formulation for photosynthetic electron transport in which the 559 cytochrome is reduced by system II and transfers electrons via the component affected in ac-21 to the 553 cytochrome. Accordingly, system I sensitizes the oxidation of the 3 components of the electron transport chain.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

Electron transport and cytochromes in aerobically grown Proteus mirabilis

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.     

Comparative receptor study in gamete chemotaxis of the seaweeds Ectocarpus siliculosus and Cutleria multifida. An approach to interspecific communication of algal gametes

The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000. This is the first report on a bacterial aa3-type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a2+(3)-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a2+(3)-NO and recombination of NO with cytochrome a2+(3) (in the 30-70 K region) revealed no differences in structure between cytochrome a2+(3) in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a2+(3)-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a2+(3)-NO. Photodissociation of cytochrome a2+(3)-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a2+(3) to cytochrome a3+. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20-40 K region) which differ completely from those observed in cytochrome a2+(3)-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a2+(3)-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu2+B-NO compound. The triplet species with delta ms = 2 EPR signals at g 4 and delta ms = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Metabolism of Pipecolic Acid in a Pseudomonas Species IV. Electron Transport Particle of Pseudomonas putida

Baginsky, Marietta L. (University of California, San Francisco Medical Center, San Francisco), and Victor W. Rodwell. Metabolism of pipecolic acid in a Pseudomonas species. IV. Electron transport particle of Pseudomonas putida. J. Bacteriol. 92:424-432. 1966.-Enzymes of Pseudomonas putida P2 catalyzing oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate are located in a subcellular fraction sedimenting at 105,000 x g. Since this fraction resembles the mammalian electron transport particle in both chemical composition and enzymatic activities, it was termed Pseudomonas P2 electron transport particle (P2-ETP). P2-ETP contains flavin adenine dinucleotide, flavin mononucleotide, iron, copper, and both b- and c-type cytochromes. The reduced type b cytochrome has absorption maxima at 558 to 559, 530, and 427 mmu. Its oxidized pyridine hemochromogen has an absorption maximum at 406 mmu, with a shoulder at 564 mmu. On dithionite reduction, absorption bands with maxima at 556, 522, and 418 mmu are obtained. The reduced type c cytochrome has absorption maxima at 552, 520, and 422 mmu; its reduced pyridine hemochromogen has maxima at 551, 516 to 519, and 418 mmu. No type a cytochrome was detected. P2-ETP catalyzes oxidation of pipecolate and of reduced nicotinamide adenine dinucleotide (NADH(2)) by oxygen. It can also oxidize these compounds, as well as succinate and reduced nicotinamide adenine dinucleotide phosphate, with 2,6-dichlorophenol-indophenol as electron acceptor. Mammalian cytochrome c can be used as an alternate artificial electron acceptor for the oxidation of pipecolate and succinate, but not for oxidation of NADH(2).     

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.     

Differences in brain cytochrome responses to carbon monoxide and cyanide in vivo

Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.     

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.     

Cytochrome components of plant microsomes.

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split-beam and dual wavelength spectrophotometry under a variety of reducing conditions. Cauliflower microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-559.5 [E'0 = +135 +/- 20 mV; lambdamax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 degrees C], cytochrome b5 [E'0 = -20 +/- 20 mV; lambdamax (reduced minus oxidised) = 556, 526 and 425 nm at 23 degrees C], cytochromes P-450 and P-420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P-450 to P-420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P-450 system is analogous to that mammalian microsomes. Other components, reducible only by dithionite, may also be present. Mung bean microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-562 [E'0 = +120 +/- 20 mV; lambdamax (reduced minus oxidised) = 562, 528 and 430 nm at 23 degrees C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P-450 or P-420 could be detected. A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.     

The electron transport system of Acetobacter suboxydans with particular reference to cytochrome o

1. The nature and distribution of the electron transport system of Acetobacter suboxydans (ATCC 621) has been investigated, with particular reference to cytochrome o.

2. A highly active membrane-bound electron transport system has been demonstrated, and functional roles suggested for ubiquinone, two c-type cytochromes ( peaks at 549 and 553 nm at — 196°), and two b-type cytochromes ( peaks at 558 and 564 nm at — 196°).

3. Evidence is presented suggesting that both the b-type cytochromes may be terminal oxidases of the cytochrome o type, and that cytochrome o (558) has an O₂ affinity approx. 10 times greater than cytochrome o (565), and a CO affinity only half as great.     

Rhein as an electron acceptor for various flavoproteins and for electron transport particles

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) which has been previously employed as an inhibitor for electron transport particles, NADH dehydrogenase, and other flavoproteins is reducible under physiological conditions. Soluble hydrogenase from Alcaligenes eutrophus H 16, several flavoproteins, and electron transport particles from baker's yeast and from beef heart were found to catalyse NADH oxidation with 9 micrometers to 2mM rhein as the electron acceptor. Dithionite or enzymatically reduced rhein (lambda max = 408 nm) is immediately reoxidized to rhein lambda max = 437 nm) by oxygen. Cyclovoltagrams reveal the midpoint redox potentials --0.240 V, -0.270 V, -0.280 V, -0.335 V at pH 6.0, 7.0, 7.7, 9.2, respectively. Due to its redox behaviour, caution should be exercised using rhein as a flavin-site-directed inhibitor for biological electron transfer systems.