Purification and properties of cytochrome c552 from purple sulfur bacterium Thiocapsa roseopersicina

The method of purification up to electrophoretical homogeneity of cytochrome c552 from the phototrophic bacterium Thiocapsa roseopersicina, strain BBS is described. For the cytochrome absorption spectrum the maxima at 417, 523 and 552 nm are characteristic for the reduced state and at 409 nm--for the oxidized state. The molecular weight is equal to 62000. The cytochrome contains two hemes per molecule and consists of two subunits. pI is 4.1; E0' is about 10 mV. Cytochrome c552 is a flavoprotein according to its fluorescence spectrum and subunit structure. T. roseopersicina cytochrome c552 is able to be reduced with sulphide, cysteine and ascorbate as well as with H2 in the presence of hydrogenase from the same bacterium. These data suggest that cytochrome c552 from T. roseopersicina functions in vivo at the initial stage of electron transport from hydrogen and sulphide.     

Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria.

Purple bacteria Rhodospirillum rubrum and Thiocapsa roseopersicina form two enzymes, hydrogenase and nitrogenase, which participate in hydrogen metabolism. H2 photoproduction in these bacteria is associated mainly or completely with the action of nitrogenase. The soluble and membrane-bound hydrogenases of T. roseopersicina have similar physicochemical properties (mol. weight, subunit composition, N-terminal amino acids, Fe2+ and S2- content, pl. Eo'). In comparison with other hydrogenases the enzyme from R. rubrum and T. roseopersicina evolve H2 with high rate from reduced cytochrome c3, but not from ferredoxins. H2 production and N2 fixation take place in the presence of NAD(P)H. NADP-reductase, ferredoxin and cytochrome c3 participate in this reaction. Possible relationships between hydrogenase-nitrogenase in the metabolism of molecular hydrogen are discussed.     

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor. Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level of cytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.     

Optical characteristics of the phototroph Thiocapsa roseopersicina and implications for real-time monitoring of the bacteriochlorophyll concentration.

Optical characteristics of a Thiocapsa roseopersicina culture and environmental samples containing T. roseopersicina were investigated in the spectral range of 400 to 1,100 nm (absorption coefficient, diffuse attenuation coefficient, and reflectance). Specific absorption coefficients of T. roseopersicina at wavelengths of 480, 520, 550, 580, 805, 860, and 880 nm were determined. It is suggested that the optical properties of T. roseopersicina in the near-infrared range of 800 to 930 nm, confirmed in this study, may be used for development of remote sensing techniques for real-time monitoring of T. roseopersicina and other bacteriochlorophyll a-containing microbes.     

Purification and properties of hydrogenase from phototrophic bacterium Thiocapsa roseopersicina]

The isolation method and some peoperties of purple sulphur bacteria (Thiocapsa roseopersicina strain BBS) hydrogenase are described Hydrogenase molecular weight is found to be 66000; it contains 3.7 moles of S2- and 3.9 moles of Fe2+ per one mole of the enzyme;pI=4.2. The enzyme absorption spectrum has the maximum at 400-412 nm which is characteristic of proteins containing non-haem iron. Hydrogenase is suggested to consist pf 4 subunits of two types: with molar weight 27000 and 6000. Unlike other hydrogenases, this enzyme is rather resistant to O2 and is more thermostable: the inactivation of the enzyme was observed at the temperature above 80 degrees C; Hydrogenase preparation catalyses D2-H2O exchange reaction, H2 evolution from the reduced methyl viologene (MV) and H2 absorption in the presense of MV or benzylviologene but not in the presense of NAD(P), FAD, FMN, azocarmine, methylene blue and ferricyanide.     

One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase

The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) [ Iyanagi , T. (1977) Biochemistry 16, 2725-2730] was confirmed to be a red anionic semiquinone. The NAD+-bound reduced enzyme was oxidized by cytochrome b5 via the semiquinone intermediate. This indicates that electron transfer from flavin to cytochrome b5 proceeds in two successive one-electron steps. Autoxidation of the NAD+-bound reduced enzyme was slower than that of the NAD+-free reduced enzyme and was accompanied by the appearance of an EPR signal. Midpoint redox potentials of the consecutive one-electron-transfer steps in the presence of excess NAD+ were Em,1 = -88 mV and Em,2 = 147 mV at pH 7.0. This corresponds to a semiquinone formation constant of 8. The values of Em,1 and Em,2 were also studied as a function of pH. A mechanism for electron transfer from NADH to cytochrome b5 is discussed on the basis of the one-electron redox potentials of the enzyme and is compared with the electron-transfer mechanism of NADPH-cytochrome P-450 reductase.     

A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.     

Comparative study of NADP-reductase properties in two species of purple bacteria]

Unlike Rhodospirillum rubrum, the highly purified preparations of NADP-reductase Thiocapsa roseopersicina are capable of reduction of cytochrome c though they do not catalyse diaphorase reaction in the presence of methyl viologen or benzyl viologen and NADH. T. roseopersicina reductase has more high temperature optimum (50-65 degrees) and more high thermal stability (65 degrees) and it is capable to catalyse diaphorase and menadione-reductase reactions under more high pH values (11.0-12.0) than NADP-reductase of R. rubrum. NADP-reductase of T. roseopersicina is more stable under storing than the enzyme from R. rubrum: the semi-inactivation period of the enzyme when storing in Ar or the air is about 10 and 4 days, respectively, and it takes about three days for R. rubrum.     

Absorption and magnetic circular dichroism spectra of hemoproteins in nonequilibrium states. V. Cytochrome P450 and its substrate complex

It was shown that ferrocytochrome P450 forms a nonequilibrium state if ferrocytochrome P450 and its complexes are reduced in freezed water-glycerol solutions by thermolysed electrons, arising during gamma-radiolysis of the matrix at 77 degrees K. Unlike the equilibrium form of ferrocytochrome P450 with the heme iron at the high-spin state the reduced nonequilibrium form of the protein contains the heme iron at a low-spin state. The absorption spectrum of ferrocytochrome P450 in the nonequilibrium state is characterized by alpha and beta-bands at 562 and 534 nm, respectively, whereas the magnetic circular dichroism spectra exhibit type A effect at 562 nm. Upon temperature increasing the nonequilibrium state is relaxed to the equilibrium one. Type 1 substrates had practically no influence on the spectral characteristic of the nonequilibrium form of ferrocytochrome P450. Binding of type 2 substrates results in an essential decrease of the intensity ratio of the alpha- and beta-bands (A alpha/A beta) and is accompanied by a red-shift of the alpha-band and corresponding magnetic circular dichroism effect. It was shown that mercaptoethanol complex of hemoglobin, formed by reduction at 77 degrees K is spectrally similar to the nonequilibrium ferrocytochrome P450 complex with type 2 substrates. From analysis of experimental data one can conclude that (i) the ligand environment of heme iron in oxidased and reduced cytochrome P450 are different; (ii) the sixth axial ligand of the heme iron in the oxidised protein is probably a water molecule (OH-) attached by a hydrogen bond to the neighbouring histidine. It is assumed that a similar nonequilibrium form of cytochrome P450 can be formed in physiological conditions.     

Spectral properties of a cyanobacterial cytochrome c oxidase: Evidence for cytochrome a.a3

Membranes isolated from $\text{Nostoc}$ sp. strain Mac oxidised NAD(P)H and horse heart ferrocytochrome c in dark reactions inhibited by KCN, NaN₃, CO, and by anaerobiosis. Reduced $\text{minus}$ oxidised difference spectra revealed peaks at 603 and 445 nm which shifted to 590 and 430 nm, respectively, in reduced $\text{plus}\text{CO}\text{minus}$ reduced spectra. In presence of suitable electron mediators the pigment could be reduced also with NAD(P)H or ascorbate; KCN prevented this reduction. Photoaction spectra of CO-inhibited membranes showed peaks at 590 and 430 nm. From the results it is concluded that cytochrome a.a₃ is a functional respiratory oxidase in $\text{Nostoc}$ sp. strain Mac.     

Competition between purple and brown phototrophic bacteria in stratified lakes: sulfide, acetate, and light as limiting factors

Abstract The affinities for sulfide and acetate under mixotrophic conditions have been determined for the brown Chlorobium phaeobacteroides and the purple Thiocapsa roseopersicina isolated from a bloom in Lake Kinneret (Israel) at a depth of about 18 m. C. phaeobacteroides exhibited a far higher affinity for sulfide than T. roseopersicina . For acetate, the opposite was observed.
In light-limited continuous cultures, C. phaeobacteroides preferentially used sulfide, whereas in mixotrophic cultures of T. roseopersicina sulfide could be detected without detectable acetate. Competition experiments under increasingly severe light limitation resulted in co-existence of the two strains. Relatively high light intensities resulted in a dominance of T. roseopersicina over C. phaeobacteroides , whereas at lower intensities C. phaeobacteroides became dominant. However, at light intensities below 2 μEin · m⁻²· s⁻¹, T. roseopersicina was completely excluded.
At low light intensities, C. phaeobacteroides is able to grow at a much higher rate than T. roseopersicina . The maintenance rate constant μ_e of C. phaeobacteroides is −0.001 h⁻¹, whereas that of T. roseopersicina is −0.011 h⁻¹. However, high light intensities inhibit the growth rate of C. phaeobacteroides , but not that of T. roseopersicina .
The explanation of the high numbers of C. phaeobacteroides in Lake Kinneret appears to be the combination of low light intensities and low sulfide concentrations. As a result, the incorporation of acetate is enhanced. The low numbers of T. roseopersicina can be explained by the high maintenance energy requirements of this organism, which exceed the available light at the depth of the bloom.     

Thiobacillus ferrooxidans cytochrome c-552: purification and some of its molecular features

Soluble cytochrome c-552 was purified from Thiobacillus ferrooxidans to an electrophoretically homogeneous state. The cytochrome showed absorption peaks at 276, 411 and 523 nm in the oxidized form and peaks at 315, 417, 523 and 552 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 13,800 on the basis of the amino acid composition and heme content, and 14,000 from SDS-polyacrylamide gel electrophoresis analysis. Its midpoint redox potential at pH 7.0 was determined to be +0.36 V. The N-terminal amino acid sequence of the cytochrome was determined as follows: A-G-G-A-G-G-P-A-P-Y-R-I-S-?-D-?-M-V-?-S-G-M-P-G-. Ferrocytochrome c-552 was oxidized by the membrane fraction of T. ferrooxidans, and the oxidation rate was more rapid at pH 3.0 than at pH 6.5. Ferricytochrome c-552 was reduced by Fe(II)-cytochrome c oxidoreductase with Fe2+ at pH 3.5, while horse ferricytochrome c was not reduced by the enzyme under the same reaction conditions.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Haem—haem interactions in cytochrome aa3 during the anaerobic-aerobic transition

A biphasic response is seen at both 445 and 605 nm as the ascorbate—cytochrome c—cytochrome aa₃ system is taken slowly from the anaerobic to the aerobic state. At low oxygen tensions the 445 nm band is more reduced while at high oxygen tensions the 605 nm band is more reduced. It is suggested that the redox potential for cytochrome a (contributing 70% at 605–630 nm and 50% at 445–455 nm) is a function of the redox state of cytochrome a₃. This model can account for both the aerobic/anaerobic data and for observations of interactions in the anaerobic system alone (Leigh, Jr, J.S., Wilson, D.F., Owen, C.S. and King, T.E. (1974) Arch. Biochem. Biophys. 160, 476–486).     

Experimental study of interactions between purple and green sulfur bacteria in sandy sediments exposed to illumination deprived of near-infrared wavelengths

Sedimentary biofilms of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404, the purple sulfur bacterium Thiocapsa roseopersicina strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-microm grain size) within counter gradients of oxygen and sulfide. The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments. A 16 h of visible light-8 h of dark regimen was used. We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm. The photosynthesis rates of P. aestuarii were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added. T. roseopersicina performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR. This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen. This way, the strict anaerobe P. aestuarii was able to grow closer to the surface in the mixed culture. As a result, P. aestuarii benefited from the presence of T. roseopersicina in the mixed culture, which was reflected by an increase in the biomass. In contrast, the density of the latter species was almost completely unaffected by the interaction. Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.     

Effect of n-octylamine on the reduction of mammalian hepatic microsomal cytochrome b5

1. In a preceding paper evidence was presented for the endogenous reduction of NAD(P)+ by mammalian hepatic microsomes and the concomitant reduction of cytochrome b5. The experiments reported here demonstrate that low concentrations of n-octylamine, in the presence of limiting quantities of NAD+, cause an increased level of cytochrome b5 reduction by mouse hepatic microsomes and also delays its reoxidation. 2. These effects are both NAD+ and n-octylamine dependent and appear to be due to an activation of the microsomal enzyme causing endogenous reduction of NAD(P)+ and also, in part, to inhibition of the autooxidation of reduced cytochrome b5. 3. Protection from the inhibitory action of sulfhydryl reagents on NADH-cytochrome b5 reductase was also observed in the presence of n-octylamine. 4. The results suggest that the enzyme(s) involved in the endogenous reduction of NAD(P)+ is not the microsomal alcohol dehydrogenase.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver.

The nicotinamide nucleotide dimers (NAD)2 and (NADP)2, obtained by electrochemical reduction of NAD+ and NADP+, are able to reduce such single-electron acceptors as the proteins cytochrome c, azurin and methaemoglobin, though at different rates. Under the same conditions the reduced nicotinamide coenzymes NADH and NADPH are not able to reduce these proteins at measurable rates unless a catalyst (phenazine methosulphate or NADH-cytochrome c reductase in the case of cytochrome) is present. The redox mechanism seems to involve the formation of an NAD(P). radical that in the presence of O2 gives rise to superoxide (O2.-), since superoxide dismutase inhibited these reactions.