Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

The cytochromes of mitochondria from Tetrahymena pyriformis strain ST

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a₆₀₃+a₃ is masked in the α-band region of spectra by the broad absorption band of cytochrome a₆₂₀. 2. Other haemoproteins present include cytochromes b₅₆₀, b₅₅₆, c₅₅₃ and c₅₄₉. 3. The reaction of reduced cytochrome a₃ with CO is reversed by flash photolysis, and in the presence of O₂ the subsequent oxidation of this cytochrome is followed by that of cytochrome a₆₀₃. 4. Cytochromes a₆₂₀ and b₅₆₀ also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a₆₀₃ to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a₆₂₀ is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.     

Light-saturated photosynthesis — Limitation by electron transport or carbon fixation?

The minimal turnover time, τ, for in vivo electron transport from water to CO₂, was calculated from oxygen flash yields and steady-state light-saturated photosynthetic rates in the marine chlorophyte, Dunaliella tertiolecta, cultured at different growth irradiance levels. As cells adapted to lower growth irradiance levels, τ increased from 3.5 to 14.5 ms, in parallel with increases in the contents of chlorophyll a, Photosystem II, PQ, cytochrome b₆f, Photosystem I and thylakoid surface density. Thus, at all growth irradiance levels examined, the relative proportion of these membrane-bound electron-transport components remained constant. However, the cellular pool size of ribulose-1,5-bisphosphate carboxylase/oxygenase, determined by radioimmunoassay, was independent of growth irradiance. Hence the ratio of the enzyme to electron-transport chain components varied between 4.8 and 1.2 as a function of growth irradiance levels. The change in this ratio was related quantitatively to the minimal turnover time of electron transport from water to carbon dioxide. Taking into account thylakoid surface density, cellular contents of electron-transport components and diffusion coefficient of plastoquinol, a diffusion time of 2.3 ms was calculated for transport of PQH₂ from Photosystem II to cytochrome b₆f. This rate is 1.5- to 13-times faster than τ. The data strongly suggest that under nutrient saturated conditions the absolute rate of light-saturated photosynthesis is limited by carbon fixation rather than electron transport. It is predicted, however, that in cells grown above 3000 μmol quanta per m² per s, electron transport rather than carbon fixation would become the rate-limiting step of light saturated photosynthesis.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

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

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

Titles of related papers in other sections

Culture of Trypanosoma cruzi (Tulahuen strain) in the presence of ethidium bromide (1–20 μg/ml) resulted in dyskinetoplasty and inhibition of growth, to an extent depending on the dye concentration and the medium composition. The ethidium bromide-induced dyskinetoplasty caused a decrease of (a) the cytochrome content of epimastigotes (a,a₃ and b species); (b) the rate of respiration (endogenous or supported by D-glucose); and (c) the rate of production of ¹⁴CO₂ from [2-¹⁴C]acetate and [1-¹⁴C]glucose. [2-¹⁴C]Acetate oxidation to ¹⁴CO₂ was affected by dyskinetoplasty more than [1-¹⁴C]glucose oxidation, particularly at the exponential growth phase. With dyskinetoplastic epimastigotes, diminution of ¹⁴CO₂ production from [2-¹⁴C]acetate largely exceeded that of oxygen uptake, while with [1-¹⁴C]glucose, ¹⁴CO₂production and respiration were affected to about the same extent. Dyskinetoplasty also decreased the incorporation of [2-¹⁴C]acetate carbon into intermediates of the tricarboxylic acid cycle and related amino acids, and modified the distribution pattern of ¹⁴C in accordance with the decrease of respiration. Reduction of cytochrome content of epimastigotes by restriction of heme compounds during growth decreased ¹⁴CO₂ production from [2-¹⁴C]acetate, like the ethidium-induced dyskinetoplasty. The same occurred after inhibition of electron transfer by antimycin and cyanide, though to a much more significant extent, thus confirming the functional association of electron transport at the mitochondrial cytochrome system of T. cruzi and the enzymatic reactions of the tricarboxylic acid cycle.     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b₆/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b₆, and Rieske Fe-S center protein in cytochrome b₆/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b₆/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b₆, and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

Salicylhydroxamic acid (SHAM) inhibits O<Subscript>2</Subscript> photoreduction which protects nitrogenase activity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. RF-1

Synechococcus sp. RF-1, a unicellular N₂-fixing cyanobacterium, can grow photosynthetically and diazotrophically in continuous light. How the organism protects its nitrogenase from damage by oxygen is unclear. In cyanobacerial cells, electron transport carriers associated with photosynthesis and respiration are all on the thylakoid membranes and share some common components, including plastoquinone pool and cytochrome b ₆ f complex, and the pathways are interacting with each other. In this work, a pulse amplitude modulation (PAM) fluorometer (PAM-101) and an O₂ electrode are used simultaneously to study the chlorophyll a fluorescence and to monitor O₂ exchanges in Synechococcus sp. RF-1 cells. At the CO₂ compensation point, the photochemical quenching activity remained high unless the O₂ was exhausted by the glucose oxidase system (GOS). It indicates that in addition to CO₂, O₂ can also act as electron acceptor to receive electrons derived from Q_A. Studies with various inhibitors of the electron transport chain demonstrated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and salicylhydroxamic acid (SHAM) inhibited the photoreduction of O₂, while glycolaldehyde, disalicylidenepropanediamine (DSPD), methyl viologen (MV) and KCN did not. These results imply that a KCN-resistant and SHAM-sensitive oxidase transfers electrons generated from Photosystem II to O₂ between cytochrome b ₆ f complex and ferredoxin. When SHAM blocked this alternative electron transport pathway, the dinitrogen-fixing activity decreased significantly. The results indicate that a novel oxidase may function as an intracellular O₂-scavenger in Synechococcus sp. RF-1 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

The electron-transport chains of the obligate methylotroph Methylophilus methylotrophus

The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O₂-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J. 192, 421–427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with α-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b₅₆₃ in methanol-limited bacteria. A third b-type cytochrome with an α-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a₃, which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O₂ in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Respiratory system of Gluconacetobacter diazotrophicus PAL5 Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases

In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q₁₀, Q₉ and PQQ in a 13:1:6.6 molar ratios. UV_360 nm photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a₁-CO adducts. Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 μM while cytochrome a₁ (442 nm) was not affected. A KCN-sensitive (I₅₀ = 5 μM) cytochrome bb and a KCN-resistant (I₅₀ = 450 μM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.     

Different contribution of rat liver microsomal pigments in the formation of superoxide anions and hydrogen peroxide during development.

Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O₂^? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b₅, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b₅ and P-450 are approximately 10%. After 20 days of development cytochrome b₅ and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O₂^? radicals have a 30% increment and reach only 60% of those of the adult; H₂O₂ production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O₂^? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O₂^? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450.     

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 Δm_s = 2 EPR signals at g 4 and Δm_s = 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.     

Reconstitution of light-dependent electron transport in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas spheroides

A mutant, O₁, of Rhodopseudomonas spheroides has been prepared that is not capable of bacteriochlorophyll synthesis, but excretes pigments spectroscopically similar to green plant chlorophylls. The cytochrome content and respiratory activity of membranes from O₁ resemble those of aerobically grown wild type R. spheroides, but the mutant could not adapt to grow photosynthetically. Photosynthetic reaction centres were purified from the blue green mutant, of R. spheroides, added to membranes from O₁, and the detergent used in reaction centre preparation removed by carefully controlled reduction. A reaction centre membrane complex was formed in which the ratio of reaction centre to cytochrome b was near 1 : 2. Illumination caused oxidation of the membrane cytochrome c and reduction of cytochrome b. These changes were enhanced in the presence of antimycin A, suggesting that a cyclic electron flow system had been reconstituted. The implication of these results on the formation of the photosynthetic electron flow system is discussed.