Juan M. Ramirez

Ferredoxin-nitrite reductase from spinach

Summary 1. Nitrite reductase from a crude homogenate of spinach leaves has been purified about 500-fold and freed from NADP-reductase (EC 1.6.99.4) and nitrate reductase (EC 1.6.6.2). The enzyme, which does not seem to be a flavoprotein, catalyzes the reduction of nitrite to ammonia with a variety of enzyme systems as electron donors and requires ferredoxin as electron carrier. Flavin nucleotides and menadione are not capable of replacing ferredoxin, but the artificial electron carrier, methyl viologen, is also effective in the reaction. In the absence of spinach NADP-reductase, ferredoxin-nitrite reductase cannot use NADPH2 as electron donor. 2 A method based on the reduction of nitrite by ferredoxin (of methyl viologen) chemically reduced with hydrosulfite has been successfully applied to the aerobic assay of the enzyme. Nitrite reductase itself is inhibited by cyanide but not by p-chloromercuribenzoate or azide. The affinities of the various substrates and the offect of pH have also been investigated. 3. Isolated spinach chloroplasts contain nitrite reductase. On a protein basis, the activity of nitrite reductase in the chloroplasts is higher than in the rest of the cell as a whole. By breaking the chloroplasts, it has been shown that most of the enzyme is recovered in the chloroplast extract and only a small part remains bound to the grana.

Flavin nucleotide nitrate reductase from spinach

Abstract 1. 1. Nitrate reductase from spinach has been purified 130-fold by a procedure which includes, as the main steps, adsorption on calcium phosphate gel and chromatography on hydroxylapatite column. By this procedure it has been shown that NADP reductase and nitrate reductase are different proteins. 2. 2. A method based on the reduction of NO3− to NO2− with flavin nucleotides reduced by S2O42− has been successfully applied to the assay of the enzyme. 3. 3. FMN and FAD are the natural cofactors which in their reduced form mediate the transfer of electrons to the nitrate-nitrate reductase system. Menadione and ferredoxin are not capable of replacing the flavin nucleotides, but benzyl and methyl viologen are affective electron carriers in the system. 4. 4. Nitrate reductase has a pH optinum of 7.6. The Michaelis constant for either FMN or FAD is roughly 0.02 mM and that for nitrate, 0.2 mM. 5. 5. Due to the fact that FMN and FAD are the effective electron donors for the reduction of NO3− in higher plants and that they can be reduced by a variety of enzyme systems, the enzyme until now known as NAD(P)H2: nitrate oxidoreductase (EC 1.6.6.2) has to be considered as a mixture of two different proteins, NADP reductase and nitrate reductase itself, and the latter will be systematically classified as FMNH2(FADH2): nitrate oxidoreductase.

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

Light and dark reduction of nitrate in a reconstituted chloroplast system

Abstract 1. 1. Reduction of NO3− by a reconstituted spinach chloroplast system both in the dark and in the light has been investigated. 2. 2. In the dark, and in the presence of FMN and nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2), NO3− can be reduced by the NADPH2-NADP reductase system. 3. 3. In the light, and in the presence of grana, FMN, and nitrate reductase, NO3− can act as the terminal electron acceptor in a new type of non-cyclic photophosphorylation. The Michaelis constant for FMN in this system is 0.02 mM. When reduced dichlorophenolindophenol is substituted for water as the electron donor, after blocking by heating the second light reaction, the rate of NO3− reduction strongly increases. This treatment also reverts the inhibition by FMN of NO2− and NADP photoreduction. 4. 4. In the light, and in the presence of grana, FMN, ferredoxin, nitrate reductase, and nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), NO3− can be successively reduced, first to NO2− and then to NH3. 5. 5. Non-cyclic photoreduction of NADP is not affected by CN− concentrations which completely inhibit the electron flow to NO3−. On the other hand, p-chloromercuribenzoate totally inhibits the photoreduction of NADP at concentrations which do not affect the nitrate system.

A possible physiological function of the oxygen-photoreducing system of Rhodospirillum rubrum.

Anaerobic suspensions of Rhodospirillum rubrum cells which had been grown in the dark under low oxygen tension showed only a small increase of their ATP content when illuminated for 30 s. The same suspensions failed to start immediate growth in the light. Both high light-induced ATP levels and immediate phototrophic growth were elicited by small amounts of oxygen which were insufficient by themselves to raise the ATP levels or to support growth in the dark. The oxygen requirement for growth disappeared after some time of anaerobic illumination and was not observed in suspensions of cells which had been grown in the light under anaerobiosis. Furthermore, these phototrophic cells reached the maximum levels of ATP when illuminated in the absence of oxygen.Strain F11, a mutant derivative of Rhodospirillum rubrum which lacked the ability to photoreduce oxygen in vitro, needed abnormally high amounts of oxygen to increase its ATP levels and to grow in the light. Besides, KCN inhibited the increase of ATP levels in illuminated mutant cells but not wild type cells. An additional difference between both strains was that the oxygen requirement for growth did not disappear in the mutant after some time of anaerobic incubation in the light.To explain these observations, it is proposed that the photosynthetic system of semiaerobically-grown Rhodospirillum rubrum becomes overreduced under anaerobiosis. The oxygen-photoreducing system, which is impaired in the mutant, is apparently used to oxidize the photosynthetic system to its optimal redox state, carrying electrons to oxygen or to other endogenous acceptors which are formed during incubation in the light. The mutant seems to replace the defective system by a cyanide-sensitive pathway which may reduce oxygen but not the alternative endogenous acceptors.

Transport of α-methyl glucoside in mutants of Escherichia coli K12 deficient in Ca2+, Mg2+-activated adenosine triphosphatase

Abstract The initial rate of uptake of methyl α-D-glucopyranoside by Escherichia coli is inhibited by respiration. The inhibition is more pronounced in mutant strains which cannot use the energy-rich state of the membrane to form ATP because of a defective Ca2+, Mg2+-activated ATPase. In both mutant and normal strains, the inhibition of glucoside uptake is not accompanied by an increase of the ATP content of the cells and is abolished by carbonyl cyanide m -chlorophenylhydrazone, a drug which dissipates membrane energy. It appears, therefore, that the inhibitory effect of respiration is mediated by the energy-rich state of the membrane and that ATP does not participate in the inhibition.

The control by respiration of the uptake of α-methyl glucoside in Escherichia coli K12

The uptake of methyl α-d-glucopyranoside (α-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells. The inhibition, which occurred only at non-saturating concentrations of α-MG, was not the result of a stimulation of the rate of exit of intracellular α-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide. Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of α-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration.

The antenna system of Rhodospirillum rubrum: Radical formation upon dark oxidation of bulk bacteriochlorophyll

11. Such near infrared changes are very similar to those observed upon light-induced or dark oxidation of photoreaction center bact- eriochlorophyll (the primary donor of bacterial photosynthesis), i.e., a bleaching at 865 nm and increase in optical density at 1245 nm [2]. Besides, the oxidized primary donor exhibits an ESR signal with a Gaussian lineshape, a g-value of 2.0025 and a peak-to-peak derivative linewidth of 9.5 G 131. In view of the similarities existing between the near infrared changes that follow oxidation of antenna and photoreaction center bacteriochlorophylls, it seemed interesting to investigate whether the oxi- dized antenna pigment is a paramagnetic species which can also be detected by ESR spectroscopy. As reported here, a nearly Gaussian ESR signal with a g-value of 2.0025 and a linewidth of 3.8 G appears to be due to the oxidized bacteriochlorophyll con- stituent which exhibits the 1230-nm band. Such a constituent seems to account only for - 1/3rd of total antenna bacteriochlorophyll and has an ap- parent midpoint redox potential of -555 mV (pH 8.0). A preliminary report of this work has been presented in [4].

Rhodoquinone as a constituent of the dark electron-transfer system of Rhodospirillum rubrum

Since 1962 Rhodospitillum rubrum has been known to contain rhodoquinone [ 11, but until [2] little was known about the specific participation of this redox carrier in the electron-transfer system of the microorganism. We showed in [2] that non-phototrophic strain Fll is a mutant deficient in rhodoquinone and that this benzoquinone is specifically required for some light-dependent redox reactions. Here we present data suggesting that rhodoquinone is also involved in 2 dark redox processes which are catalyzed by membrane vesicles isolated from R. rubrum, the reduction of tetrazolium blue by succinate and that of fumarate by NADH. It seems possible that both reactions are mediated by the same catalytic system.

A specific role for rhodoquinone in the photosynthetic electron-transfer system of Rhodospirillum rubrum

Quinones are essential constituents of the electrontransfer systems which mediate energy conversion in biological membranes. The functional versatility of these lipophilic redox agents is particularly apparent in bacterial photosynthesis, where quinones appear to participate in at least 4 distinct redox steps [ 11. In the facultative photoheterotroph Rhodospirillum rubrum, all those functions have been ascribed to ubiquinone-IO [2] whereas no specific participation in photochemical electron transfer has been demonstrated for rhodoquinone-10, the other quinone detected in the membrane system of that bacterium [3]. Here we describe the characterization of a non-phototrophic R. rubrum mutant which lacks rhodoquinone. The properties of this strain indicate that rhodoquinone is specifically required for R. rubrum photosynthesis as a constituent of the electron-transfer side chain which is apparently involved in the redox regulation of the main cyclic pathway [4-61.