Evidence for one-electron transfer by the Fe protein of nitrogenase

The number of electrons transferred per molecule of the Fe protein of nitrogenase from $\text{Clostridium pasteurianum}$ was determined. The Fe protein was enzymically oxidized in the presence of MgATP and a small amount of MoFe protein, and dithionite was introduced to reduce part of the Fe protein. From the decrease in absorbance at 430 nm upon addition of dithionite and the amount of dithionite added, we conclude that one oxidized Fe protein molecule (dimer of 55,000 dalton) accepts one electron from dithionite. These calculations were based on our value of 6,600 M^?1cm^?1 for the extinction coefficient at 430 nm of the difference spectrum between oxidized and reduced Fe protein.     

A cobalt containing protein isolated from Desulfovibrio gigas, a sulfate reducer

A protein which contains a cobalt porphyrin was isolated from the sulfate reducer Desulfovibrio gigas. This protein has a molecular weight of approximately 16,700 daltons and is acidic, having an iso-electric point at 3.7. The N-terminal residue was shown to be threonine, and a cobalt analysis gave 0.8 cobalt atoms/molecule, suggesting the presence of a single prosthetic group. The protein has a violet color with absorption bands typical of a metal porphyrin center with maxima at 420 nm, 580 nm with a shoulder at 550 nm. The ratio $\text{A}{}_{420}\text{(γ)}\text{A}{}_{588}\text{(α)}$ is 2.1. The protein has no electron paramagnetic resonance (e.p.r.) spectrum, and as the visible spectrum suggests, it probably contains diamagnetic Co^III porphyrin. However the cobalt centre appears to be protected from reduction by sodium dithionite or sodium borohydride. Att$\text{e}$mpts at ligand substitution with strong nucleophiles such as CN, causes a slight spectral shift to higher wavelenghts. The cobalt porphyrin can be extracted from the protein with an acidified acetone solution, indicating that it is not covalently bound to the protein.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Adenocarcinoma of the human exocrine pancreas: presence of secretin and caerulein receptors

We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome $\text{b}$-559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes $\text{f}\text{,}\text{b}$-563 and $\text{b}$-559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems.Cytochrome $\text{f}$ was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome $\text{b}$-563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

Electron transport components from methanogenic bacteria: The ferredoxin from Methanosarcinabarkeri (strain Fusaro)

A ferredoxin has been isolated from the methanogenic organism $\text{Methanosarcina}\text{barkeri}$ (strain Fusaro). The protein appears to be constituted by two identical subunits of molecular weight approx. 6000 daltons. The UV-visible spectrum of the protein is characterized by two broad absorption peaks centered at 410 and 300 nm and an absorbance ratio $\text{A}{}_{410}\text{A}{}_{300}\text{= 0.8}$. The molar extinction coefficients at 410 and 300 nm are 36,500 and 45,625 M^?1 cm^?1, respectively. The amino acid compsition of $\text{M.}\text{barkeri}$ ferredoxin shows a preponderance of acidic residues and lacks five amino acids. The protein contains 8 cysteine residues and approx. 7 iron atoms and 7–8 acid-labile sulfide groups per molecule which are indicative of the presence of two iron-sulfur clusters in the molecule. The N-terminal sequence shows a high degree of homology with the sequences of ferredoxins from $\text{Clostridium}\text{pasteurianum}\text{,}\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{africanus}\text{.}\text{M.}\text{barkeri}$ ferredoxin functions as an electron carrier in the pyruvate dehydrogenase system. Its possible role in a variety of electron transfer reactions is discussed.     

Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles

Phagocytic vacuoles containing lgG coated latex particles were isolated from human neutrophils by floatation. The absorbance spectrum of the cytochrome $\text{b}$ was associated with the vacuoles within 10 sec of particle uptake and the vacuolar concentration increased little thereafter. In contrast, the cytoplasmic granule proteins myeloperoxidase and vitamin B₁₂ binding protein associate with the vacuoles more slowly. The addition of dithionite to intact cells rapidly reduces most of the cytochrome $\text{b}$, whereas only a small proportion of the myeloperoxidase, which is located intracellularly, is reduced in the absence of detergent. Most of the cytochrome $\text{b}$ appears to be localised in the neutrophil plasma membrane.     

Evidence for water as the product for oxygen reduction by cytochrome cd.

Evidence is presented that water is the final product of electron donation to molecular oxygen by cytochrome $\text{cd}$ from $\text{Paracoccus}\text{denitrificans}$ when ferrocytochrome $\text{c}$ acts as donor to $\text{cd}$. Negative evidence for the accumulation of superoxide and peroxide was obtained by rate effect experiments in the presence of superoxide dismutase, catalase, and peroxidase. Positive evidence for water was obtained by showing a 4 to 1 stoichiometric balance for rates of electron acceptance from ferrocytochrome $\text{c}$ to rates of donation to molecular oxygen.     

Preparation of pyridoxal 5'-phosphate-bound sepharose and its use for immobilization of tryptophanase

Pyridoxal 5′-phosphate-bound Sepharose (SP) was prepared by coupling pyridoxal 5′-phosphate (PLP) to diazotized p-aminobenzamidohexyl-Sepharose. A derivative of pyridoxine having an absorption maximum at $\text{ca.}$ 316 nm (possibly, 6-amino-pyridoxine 5′-phosphate) was liberated from SP by treatment with 0.1 M sodium dithionite at pH 9.0. SP catalyzed the cleavage of tryptophan in the presence of Cu²⁺, a typical non-enzymatic model of tryptophanase reaction. From the spectrophotometric data and catalytic activity, it was estimated that SP contained about 1.5 μmoles of bound PLP per gram of Sepharose. Tetrameric apotryptophanase was immobilized by incubation with SP, followed by reduction with NaBH₄. The resulting immobilized tryptophanase retained $\text{ca.}$ 60 % of the catalytic activity of free tryptophanase used. This method was much superior to other methods used commonly for preparation of immobilized enzymes.     

Presence and nature of a glycocalyx-like coat on the external vesicular membrane of cysticercus cellulosae. A high resolution histochemical study.

Three histochemical techniques were used to demonstrate the presence of mucosubstances on the exposed surface of the vesicular membrane of $\text{Cysticercus cellulosae.}$ Using Alcian blue staining, electron micrographs revealed electron dense deposits of lanthanum confined to the entire exposed surface of the microthriches. Using concanavalin A, the results showed a discontinuous thin layer of electron dense deposits along the same exposed surface as the Alcian blue technique. Using colloidal iron hydroxide labelling, the electron micrographs showed the presence of negative charges along only the tips of the microthriches. This distribution of the negative charges is discussed and possible explanations are proposed. The conclusion from these results on both the presence of a surface glycocalyx-like coat rich in both acidic and neutral carbohydrates and the presence of a negative surface potential suggest that the exposed surface of $\text{Cysticercus cellulosae}$ may have important roles in the host-parasite interrelationships.     

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700⁺ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 μs back reaction of P-700⁺, which we associate with a more primary electron acceptor A₂. In turn, when A₂ is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 μs) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A₁, which functions very close to P-700. The organization of these components probably corresponds to the sequence $\text{P-700-}\text{A}{}_1\text{-}\text{A}{}_2\text{-P-430[}\text{A}\text{B}\text{]}$. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed.Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

A Novel function of cytochrome C (555, Chlorobium thiosulfatophilum) in oxidation of thiosulfate

Thiosulfate-cytochrome c-551 reductase derived from $\text{Chlorobium}\text{thiosulfatophilum}$ has been highly purified. The enzyme reduces cytochrome $\text{c}\text{-551 of}\text{C}\text{.}\text{thiosulfatophilum}$ in the presence of thiosulfate while cytochrome $\text{c}$-555 of the organism is not reduced by the enzyme. Cytochrome $\text{c}$-555 reacts with the enzyme at an appreciable rate only in the presence of cytochrome $\text{c}$-551. However, the reduction rate of cytochrome $\text{c}$-551 by the enzyme is greatly enhanced on addition of a catalytic amount of cytochrome $\text{c}$-555. Therefore, cytochrome $\text{c}$-555 seems to function as an effector on thiosulfate-cytochrome $\text{c}$-551 reductase as well as it acts as the electron donor to the light-excited chlorobium chlorophylls.     

Pyruvate-supported acetylene and sulfate reduction by cell-free extracts of Desulfovibrio desulfricans

Cell-free extracts prepared from a strain of $\text{Desulfovibrio}\text{desulfricans}$ can reduce acetylene or sulfate, utilizing pyruvate as the electron and ATP source in the presence of methyl viologen, ADP and coenzyme A. Other physiological substances such as (lactate + NAD) and (NADH + ATP) can not reduce acetylene nor sulfate. When acetylene and sulfate are both present as substrates, sulfate represses the acetylene reduction.     

Quantum requirements for proton uptake in spinach chloroplasts

Studies of electron and proton transport in chloroplast preparations (Type D) from spinach (Spinacea oleracea L.) yield three basic results. First, in electron transport catalyzed by methyl viologen from water to oxygen at pH 7.6, the quantum requirement for electron transport ($\text{hv}\text{e}{}^{\mathrm{?}}$) was 2.2, while the corresponding requirement for proton transport ($\text{h}\text{v}\text{H}{}^{\mathrm{+}}$) was 1.2. Second, the electron and proton quantum requirements were relatively independent of the individual chloroplast preparation or certain components of the resuspension medium, but did depend upon the reaction medium's initial pH. Third, measurable electron and proton transport did not occur under 715-nm illumination, nor did such activities occur in the presence of DCMU under 645-nm illumination when methyl viologen was used as the electron transport cofactor. These experimental results reconcile the quantum requirement of proton transport with Mitchell's chemiosmotic theory for chloroplast energy transduction and resolve a long standing controversy regarding the quantum requirement in chloroplast thylakoids.     

Evidence for a protonmotive Q cycle mechanism of electron transfer through the cytochrome b-c1 complex.

A protein named oxidation factor can be reversibly removed from succinate-cytochrome $\text{c}$ reductase complex and shown to be required for electron transfer between succinate and cytochrome $\text{c}$. This protein is required for reduction of cytochrome $\text{c}{}_1$ and, in the presence of antimycin, for reduction of both cytochromes $\text{b}$ and $\text{c}{}_1$. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome $\text{c}{}_1$ and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome $\text{b}\text{-}\text{c}{}_1$ complex.     

Purification of the agent inducing lipid peroxidation with dihydroxyfumaric acid in rat liver mitochondria

Dihydroxyfumaric acid induces lipid peroxidation in rat liver mitochondria as reported previously. When the mitochondria were solubilized with 0.35% ($\text{W}\text{V}$) sodium cholate, the supernatant itself could not catalyze lipid peroxidation with dihydroxyfumaric acid, but the precipitate slightly induced the reaction. The supernatant produced lipid peroxide in the presence of the precipitate and dihydroxyfumaric acid. The supernatant was heat sensitive contrary to the stability of the precipitate. An attempt was made to isolate active entity through a sephadex G-200 column and a DEAE-cellulose column, resulting in about 10-fold purification. At 408–410 nm the partially purified agent showed a maximum absorption, which disappeared rapidly after reduction with sodium dithionite and was slowly diminished with dihydroxyfumaric acid. The molecular weight was much larger than that of oxidized cytochrome c.