Electron Transport System Associated with Membranes of Bacillus cereus During Vegetative Growth and Sporulation

Membranes isolated from Bacillus cereus ATCC 4342 during vegetative growth and during sporulation contained cytochromes b, c and a + a(3) as well as flavoprotein as determined from reduced-minus-oxidized difference spectra. Although there appeared to be no qualitative change in the cytochromes, there was a significant increase in the amount of cytochromes associated with membranes isolated from sporulating cells. Succinate and nicotinamide adenine dinucleotide (reduced form) (NADH) reduced the same cytochromes indicating similar pathways of electron transport. The electron transport inhibitors-cyanide, azide, 2-heptyl-4-hydroxyquinoline-N-oxide, dicumarol and atebrine-were examined for their effect on succinate oxidase (succinate: [O(2)] oxidoreductase) and NADH oxidase (NADH: [O(2)] oxidoreductase). NADH oxidase associated with vegetative cell membranes was less sensitive to certain inhibitors than was succinate oxidase, suggesting a branched electron transport pathway for NADH oxidation. In addition to electrons being passed to O(2) through a quinone-cytochrome chain, it appears that these intermediate carriers can be bypassed such that O(2) is reduced by electrons mediated by NADH dehydrogenase. Both oxidases associated with sporulating cell membranes were inhibited to a lesser degree than were the oxidases associated with vegetative cell membranes.     

Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM.

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.     

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM.

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a3, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a3, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N',N'-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.     

Oxidation of sulfur compounds and electron transport in Thiobacillus denitrificans

Summary Intact cells of Thiobacillus denitrificans catalyzed the oxidation of thiosulfate, sulfide and sulfite with nitrate or oxygen as the terminal acceptor. The anaerobic oxidation of thiosulfate, sulfide and sulfite was sensitive to the inhibitors of the flavoprotein system. Under aerobic conditions the oxidation of sulfide and sulfite was sensitive to these inhibitors but the thiosulfate oxidation was unaffected. Cyanide and azide inhibited the aerobic and anaerobic respiration when thiosulfate, sulfide or sulfite served as electron donors. The oxidation of thiosulfate by cell-free preparations was mediated by cytochromes of c, a and o-types. The cell-free extracts also catalyzed the oxidation of NADH and succinate, involving flavoproteins and b, c, a and o-type cytochromes. In addition, a cytochrome oxidase sensitive to cyanide and azide was also present.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - HQNO 2-heptyl-4-hydroxyquonoline N-oxide Aspirant van het Nationaal Fonds voor Wetenschappelijk Onderzoek (Belgian National Science Foundation).     

Electron transport in a Park-Williams strain of Corynebacterium diphtheriae

1. The electron-transport mechanism was examined in the ;particulate' and ;supernatant' fractions of disintegrated cells of a Park-Williams strain of Corynebacterium diphtheriae. 2. Succinate-oxidase activity was found mainly in the ;particulate' fraction, and NADH(2) oxidase mainly in the ;supernatant', which was devoid of cytochromes and menaquinone. 3. The sum of the activities of particles and supernatant fractions, with respect to both succinate oxidase and NADH(2) oxidase, was substantially less than that of the crude cell extract from which they were obtained. Full activity was restored on recombining ;particles' and ;supernatant'. The characteristics of this reassembled system were investigated. 4. The strain of organism (CN2000) examined contained cytochromes corresponding spectroscopically to ;a', ;b' and ;c' types. All three were reduced by succinate, lactate or NADH(2); but a portion of the cytochrome b, susceptible to reduction by dithionite, could not be reduced by the substrates. 5. Triton X-100 inhibits oxidation of succinate by particulate fraction; on adding succinate, the reduction of cytochrome b is not affected but that of cytochromes a and c is delayed. 6. Irradiation at 360mmu completely destroys menaquinone in the particle fraction. Succinate oxidation is severely decreased; succinate dehydrogenase and NADH(2) oxidation are little affected. Certain menaquinones will restore succinate oxidation in the irradiated material. 7. On adding succinate to irradiated particulate material cytochrome b is partially reduced at once, but reduction of cytochromes a and c is much delayed. A portion of the cytochrome b remains not reduced, but reduction occurs rapidly on the addition of menaquinone (MK-2).     

Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system.

Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Blue light-sensitive plasma membrane bound exogenous NADH oxidase in Cuscuta reflexa

Protoplasts isolated from Cuscuta reflexa exhibited a higher rate of exogenous NADH oxidation as compared to NADPH in the dark. NAD(P)H oxidation was monitored by measuring the rate of oxygen consumption and this oxidase system was sensitive to blue light. Both NADH oxidase and its blue light sensitivity were inhibited by -SH group reacting agents. The corresponding changes occurring in H+-extrusion activity and intracellular ATP levels were also monitored. Stimulation of NADH oxidation under blue light corresponded to increased rate of H+-extrusion and intracellular ATP level, the converse was also true under NADH oxidase inhibitory conditions. These observations suggested a close functional association between blue light-sensitive plasma membrane bound redox activity and H+-ATPase in this tissue. Further, concanavalin A binding of protoplasts resulted in a loss in NADH oxidase activity and its blue light sensitivity suggesting apoplastic location and glycoprotein nature of the blue light sensitive NADH oxidase system in Cuscuta.     

Carbon monoxide-insensitive respiratory chain of Pseudomonas carboxydovorans.

Experiments employing electron transport inhibitors, room- and low-temperature spectroscopy, and photochemical action spectra have led to a model for the respiratory chain of Pseudomonas carboxydovorans. The chain is branched at the level of b-type cytochromes or ubiquinone. One branch (heterotrophic branch) contained cytochromes b558, c, and a1; the second branch (autotrophic branch) allowed growth in the presence of CO and contained cytochromes b561 and o (b563). Electrons from the oxidation of organic substrates were predominantly channelled into the heterotrophic branch, whereas electrons derived from the oxidation of CO or H2 could use both branches. Tetramethyl-p-phenylenediamine was oxidized via cytochromes c and a exclusively. The heterotrophic branch was sensitive to antimycin A, CO, and micromolar concentrations of cyanide. The autotrophic branch was sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide, insensitive to CO, and inhibited only by millimolar concentrations of cyanide. The functioning of cytochrome a1 as a terminal oxidase was established by photochemical action spectra. Reoxidation experiments established the functioning of cytochrome o as an alternative CO-insensitive terminal oxidase of the autotrophic branch.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

The microbial metabolism of C1 compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.     

Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.     

NADH: quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus.

The site of Na+-dependent activation in the respiratory chain of the marine bacterium, Vibrio alginolyticus, was investigated. The respiratory chain system contained ubiquinones (Q), menaquinones (MK), cytochromes b(560), c(553), d(630), and o(560). The membrane-bound and partially purified NADH dehydrogenase was stimulated 2- to 3-fold by the addition of 0.2 M Na+ or K+ and no specific requirement for Na+ was observed in this reaction step. The cytochrome oxidase showed no requirement for monovalent cations. The respiratory activity (NADH oxidase) of the membrane was lost on removal of the quinones, and the reincorporation of authentic Q-10 or MK-4 restored the activity. The rate of MK-4 reduction by NADH (menaquinone reductase) as measured using MK-4 incorporated membrane was activated by Na+, but only slightly by K+. The apparent Ka for Na+ was 78 mM for both menaguinone reductase and NADH oxidase. The requirement for Na+ of menaquinone reductase was greatly reduced in the presence of 0.2 M K+. Ubiquinone reductase as measured by using Q-10 incorporated membrane was also activated more effectively by Na+ than by K+. These results strongly suggested that the site of Na+-dependent activation in the respiratory chain of marine V. alginolyticus was at the step of NADH; quinone oxidoreductase.     

The activity of tissue enzymes in iron-deficient rat and man: an overview

The effects of iron deficiency in rat and/or man on iron-containing enzymes of different tissues is reviewed. Iron deficiency results in a decrease of skeletal muscle iron containing proteins e.g. myoglobin, cytochromes c, a + a3, and alpha-glycerophosphate oxidase. Iron deficiency produces a reduction in the activity of several respiratory enzymes in the mitochondrial fraction of cardiac muscle, particularly: NADH cytochrome c reductase, succinic cytochrome c reductase, succinic dehydrogenase and NADH ferricyanide oxidoreductase. The effects of iron deficiency on brain tissue is emphasized with respect to cytochromes, monoaminoxidase and amino acids metabolism. Host defence to infection (controversial data), decrease in body temperature, alteration of DNA synthesis, collagen and lipid metabolism, liver and gastrointestinal mucous cytochromes activity perturbations are discussed.     

Alteration of inner-membrane components and damage to electron-transfer activities of bovine heart submitochondrial particles induced by NADPH-dependent lipid peroxidation.

We investigated the changes of the inner-membrane components and the electron-transfer activities of bovine heart submitochondrial particles induced by the lipid peroxidation supported by NADPH in the presence of ADP-Fe3+. Most of the polyunsaturated fatty acids were lost as a result of the peroxidation, and phospholipids were changed to polar species. Ubiquinone was also modified to polar substances as the peroxidation proceeded. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the disappearance of 27000-Mr and 30000-Mr proteins and the appearance of highly polymerized substances. Flavins and cytochromes were not diminished, but the respiratory activity was lost. The reactions of NADH oxidase and NADH-cytochrome c reductase were most sensitive to the peroxidation, followed by those of succinate oxidase and succinate-cytochrome c reductase. Succinate dehydrogenase and duroquinol-cytochrome c reductase were inactivated by more extensive peroxidation, but cytochrome c oxidase was only partially inactivated. NADH-ferricyanide reductase was not inactivated. The pattern of the inactivation indicated that the lipid peroxidation affected the electron transport intensively between NADH dehydrogenase and ubiquinone, and moderately at the succinate dehydrogenase step and between ubiquinone and cytochrome c.     

The respiratory system of Pseudomonas putida: participation of cytochromes in electron transport

Rates of oxygen utilization by Pseudomonas putida respiratory particles were measured using the electron donors, reduced nicotinamide adenine dinucleotide (NADH) and succinate, and the oxidation-reduction dyes, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethyl-p-phenylenediamine. The maximal rates produced by NADH and succinate were similar for particles from either log- or stationary-phase cells, but rates measured using the dyes were much higher in stationary-phase particles. Cyanide and azide were very effective inhibitors of dye oxidation in both cases, but they produced only partial inhibition of NADH and succinate oxidation in log-phase particles and had no effect in the stationary phase. Spectral examination of the cytochromes at several levels of reduction produced by the various electron donors and inhibitors indicated that most of the cytochromes that were reduced by the dyes lie on a cyanide sensitive pathway of electron transport. These findings support the hypothesis that P. putida produces an electron transport system in the stationary phase which involves branching at the level of the cytochromes.Inhibition of oxygen utilization by CO was nearly complete for all four substrates in logphase particles. Inhibition was also reasonably effective for dye oxidation in the stationary phase, but there was no effect on NADH or succinate oxidation. Photochemical action spectra of the relief of CO inhibition revealed that NADH and succinate oxidation in log-phase particles probably involves cytochrome o. Oxidation of the dyes by either type of particles also appeared to involve cytochrome o, and the possibility of the participation of an a- or d-type cytochrome was also indicated.     

The role of ubiquinone in the respiratory chain of Acetobacter xylinum

1. Whole cells of Acetobacter xylinum were found to contain a quinone of the ubiquinone (coenzyme Q) group. The quinone was isolated from the cells and crystallized. It was identified by its physical, chemical and spectroscopic properties as a ubiquinone with 10 isoprene units (ubiquinone-10). No naphthaquinone was detected in the cells. 2. Cell-free extracts prepared by means of a French pressure cell were separated into three fractions by differential centrifugation. The ubiquinone was located predominantly in the particulate fraction sedimenting at 33000g, which also contained most of the NADH oxidase and malate oxidase activities. The concentration of ubiquinone-10 in extracts was similar to that of the flavoproteins and about three times the concentration of the individual cytochromes. 3. Aerobic incubations of crude extracts with either NADH or malate resulted in reduction of the endogenous ubiquinone-10 to steady-state concentrations of 55 and 40% of the total quinone respectively. In the presence of cyanide more than 95% of the endogenous ubiquinone-10 was reduced by either NADH or malate. 4. The initial rate of reduction of endogenous ubiquinone-10 by malate and the rate of ubiquinol oxidation, in A. xylinum extracts, were found to be compatible with the overall rate of malate oxidation with oxygen. 5. The effects of various respiratory inhibitors on the oxidation-reduction reactions of the endogenous quinone indicate that its position on the respiratory chain is between the malate flavoprotein dehydrogenase and the cytochrome chain.