Effects of selected inhibitors on electron transport in Neisseria gonorrhoeae.

The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation of reduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340 nm, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytal-rotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbate-tetramethyl-p-phenylenediamine suggest the participation of b-, a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d-type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component.     

Cytochrome pools in membranes of Escherichia coli grown aerobically on L-proline

The cytochromes of membranes of the cydA mutant Escherichia coli GR19N grown on a proline-amino acid medium were examined. Reduced minus oxidized difference spectra (including fourth-order finite difference spectra) showed that cytochromes with absorption maxima at 554-555, 556-557, 560-561.5 and 563.5-564.5 nm were present. In addition, there were two components with absorption maxima at 548.5 and 551.5 nm which made a minor contribution to the alpha-band absorbance. These were not examined further. Two pools within the cytochromes were detected. One pool, which was reduced rapidly by the substrates NADH, formate and succinate, consisted of cytochromes of the cytochrome o complex. These cytochromes had absorption maxima at 555, 557 and 563.5 nm. In addition, the low-potential cytochrome associated with formate dehydrogenase was reduced rapidly by formate, and a component absorbing at 560-561.5 nm was also present in this pool. The second pool of cytochromes was reduced more slowly by substrate, although the rate was accelerated greatly in the presence of the electron mediator phenazine methosulfate. These cytochromes absorbed maximally at about 556.5 nm. A portion of the cytochrome in this pool was reoxidized by fumarate. This cytochrome may be a component of the fumarate reductase pathway, since the membranes showed high NADH-fumarate reductase activity. The respiratory chain inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide appeared to act at two sites. One site of inhibition was between the dehydrogenases and the cytochromes. A second site of inhibition was located in the cytochrome o complex between cytochrome b-564 and oxygen.     

Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes

In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes. The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome. This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B. subtilis. The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature. The oxidized cytochrome had an absorption maximum at 413 nm. The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes. In whole membranes cytochromes b, c, and a were reduced by succinate. In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate. It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B. subtilis membrane. An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.     

The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris.

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically growth Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three beta-types cytochromes b561, b560 and b558, and at least two c-type cytochromes c556 and c2 as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b560, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c'. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b561 with associated beta and gamma bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c2 may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Oxidation-reduction potentials and absorption spectra of two b-type cytochromes from the halophilic archaebacterium, Halobacterium halobium

The oxidation-reduction midpoint potentials were determined for two b-type cytochromes, which had been solubilized from the membrane of Halobacterium halobium and partially purified. The two b-type cytochromes have oxidation-reduction midpoint potentials of 175 and 7 mV, respectively. These b-type cytochromes could also be resolved by difference absorption spectroscopy, which revealed one b-type cytochrome with absorption maximum (alpha-peak) at 558 nm, reducible by ascorbate-tetramethyl-p-phenylenediamine, and the other with absorption maximum (alpha-peak) at 560 nm, reducible by dithionite. Different substrates such as succinate, NADH, and alpha-glycerophosphate were used to study the b-type cytochromes in situ when bound to the membrane in a functional state. Reducing equivalents from succinate and alpha-glycerophosphate appear to enter the respiratory chain at the 175 mV b-type cytochrome. Cytochrome a3 is spectrophotometrically shown to be present in the membrane of H. halobium.     

Cytochromes and hydrogen-oxidizing activity in the thermophilic hydrogen-oxidizing bacteria related to the genus Hydrogenobacter

The highly thermophilic, hydrogen-oxidizing aerobic bacteria related to Hydrogenobacter possess a respiratory chain comprising a quinone and b-type (alpha band at 556 nm and 562 nm) and c-type (alpha band at 552 nm) cytochromes. They have no aa₃-type cytochromes and their terminal oxidase is an o-type cytochrome. A polarographic method with an oxygen electrode was used for the measurement of the hydrogen-oxidizing activity. This activity was strongly inhibited by HQNO (2-N-heptyl-4-hydroxyquinoline N-oxide), an inhibitor of the respiratory chain in the quinone-cytochrome b region, and by KCN, an inhibitor of the terminal cytochrome oxidase. This study shows that the electrons released from hydrogen oxidation by the membrane-bound hydrogenase probably enter the respiratory chain at the level of the quinone-cytochrome b region.Abbreviations HQNO 2-N-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine - DW dry weight     

Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria

The free radical EPR signals of ubisemiquinone in mitochondria and submitochondrial particles (SMP) were investigated. One of the signals observed under the conditions of the respiratory chain highly oxidized and characterized by an unusually short time of the spin-lattice relaxation has previously been termed as SQ-2. The intensity of SQ-2 in SMP strongly depends on pH, the maximal concentration of QH. is reached at about 8.5. The signal is absent in the succinate dehydrogenase-depleted SMP and is highly sensitive to specific inhibitors of succinate: CoQ-oxidoreductase, such as alpha-thenoyltrifluoroacetone and carboxin. In SMP SQ-2 disappears in the presence of low concentrations of ferricyanide, while in mitochondria this non-penetrating oxidant provokes the appearance of SQ-2. The data obtained suggest that SQ-2 belongs to a stable ubisemiquinone which forms a complex with a FeS center of succinate dehydrogenase, is localized at the M-side of the membrane, and is kinetically isolated from the cytochrome chain. Oxidation of the terminal segment of the respiratory chain of mitochondria and SMP reduced by succinate in the presence of antimycin, is in some cases accompanied by an appearance of a strong free radical EPR signal which is stable at 77K but disappears rapidly in the frozen samples at -30- -40 degrees C. It is suggested that the signal is generated by an antimycin-insensitive oxidation of QH2 to QH. via the branch of the respiratory chain comprised of the Rieske FeS-protein and cytochrome c1. The mechanisms of how the two-electron oxidation-reduction of CoQ is coupled with the one-electron transfer through the cytochromes and FeS centers in the respiratory chain are discussed.     

Spectral properties of cytochromes from Staphylococcus aureus

Two cytochrome b with peaks at 554 and 558 nm and cytochrome a with alpha-peak at 603 nm were found in intact cells and membranes of Staphylococcus aureus using low-temperature spectrophotometry and registration of second- and fourth-order finite difference spectra of cytochromes. Analysis of the cytochrome functioning in membranes isolated from the cells at the exponential and stationary growth phases revealed no difference in the set of these carriers. Analysis of cytochrome reduction with different substrates demonstrated identity of the cytochrome composition in the respiratory chain, reduced with NADH, lactate, alpha-glycerophosphate, malate and succinate. Cytochrome omicron with gamma-peak at 416 nm in the CO-spectra was found to be involved in oxidation of all the substrates tested both in intact cells and membranes of Staphylococcus aureus.     

Effects of ethanol and acetaldehyde on iron-sulfure centers in the mitochondrial respiratory chain.

Incubation of submitochondrial particles with relatively low concentrations of ethanol (20–100 mm) or acetaldehyde (1–10 mm) produces alterations in the electron paramagnetic resonance spectra of the iron-sulfur centers in the NADH dehydrogenase segments of the respiratory chain. The iron-sulfur centers in the NADH dehydrogenase region are most sensitive to both ethanol and acetaldehyde, in comparison to the iron-sulfur centers in succinate dehydrogenase and the cytochrome b-c region. Centers N-3, 4, N-5, 6 and N-1b are affected after relatively short incubation periods (3–30 min) while center N-2 shows considerable sensitivity over somewhat longer incubations (20–90 min). The most ethanol-sensitive center in the succinate dehydrogenase region of the respiratory chain is high potential iron-sulfur protein-type center S-3. Potentiometric analysis shows that these alterations are not due to simple changes in the redox state caused by addition of dissolved oxygen. Changes in the electron paramagnetic resonance spectra can be correlated with decreased rates of oxidation of NADH and, to a lesser extent, succinate in both ethanol- and acetaldehyde-treated submitochondrial particles.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart.

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).     

Respiration capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs

Mitochondria were isolated from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus purpuratus. Both preparations exhibited coupled adenosine 5'-diphosphate (ADP)-dependent) oxidation of flavin and pyridine-linked substrates and both yielded the expected P:O ratios with these substrates. Highest respiratory control indices (greater than 4.0) were observed when succinate or pyruvate + malate were used as substrates. Mitochondria from unfertilized and fertilized eggs exhibited sensitivity to respiratory and phosphorylation inhibitors and uncouplers and both preparations exhibited cross-over points at sites I, II and III of the respiratory chain. Low-temperature difference spectra revealed a normal complement of cytochromes c, b and aa3, although cytochrome c from unfertilized eggs appears to be more subject to extraction during the course of mitochondrial isolation than does cytochrome c from fertilized eggs. An unidentified pigment absorbing at approx. 570 nm was visible in low-temperature spectra of unfertilized eggs and unfertilized egg mitochondria.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : V. On the Reduction of Nonheme Iron and the Cytochromes by Nicotinamide Adenine Dinucleotide and Succinate

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.     

Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes

Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.     

Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species

Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome.     

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy

We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten-halogen light source. Emergent light in the visible region (from 490-650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a 'steady-state' baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.     

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.     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.