The hydroxylation of P-cresol and its conversion to P-hydroxybenzaldehyde in Pseudomonas putida.

Cell extracts of $\text{Pseudomonas putida}$ catalyze the conversion of $\text{p}$-cresol to $\text{p}$-hydroxybenzylalcohol when phenazine methosulfate, an electron acceptor, is added. The reaction will proceed under anaerobic conditions and a mechanism involving dehydrogenation to a heteroquinone followed by hydration is proposed. This contrasts with the known attack on methyl groups by mono-oxygenases. The same requirements are found for the alcohol dehydrogenase and the major product from reaction mixtures is the aldehyde. Of the compounds tested as substrates only those with the appropriate groups in the $\text{para}$ orientation were attacked.     

The interaction between the heme c and heme d moieties of Pseudomonas nitrite reductase as revealed by magnetic and natural circular dichroism studies.

A possibility of a heme-heme interaction between the heme $\text{c}$ and heme $\text{d}$ moieties in $\text{Pseudomonas}$ nitrite reductase was examined by using magnetic and natural circular dichroism. The MCD of the heme $\text{c}$ moiety in the ferric enzyme was similar to that of mammalian ferricytochrome $\text{c}$ in shape and intensity, whereas in the reduced state the MCD intensity was considerably smaller than that of ferrocytochrome $\text{c}$. When the heme $\text{d}$ moiety was perturbed by the complex formation with CO, imidazole or cyanide as well as by pH changes, the depressed MCD was restored to the MCD level of mammalian ferrocytochrome $\text{c}$, accompanying conformational changes around the prosthetic groups. Thus, it was concluded that the heme-heme interaction exists only in the reduced enzyme and that this interaction is released under appropriate conditions.     

Biological activity of synthetic tetranuclear iron-sulphur analogues of the active sites of ferredoxins.

Two water-soluble tetranuclear iron-sulphur clusters have been synthesised which are stable under anaerobic conditions in the presence of excess thiol in aqueous solution. In such a solution, in the presence of sodium dithionite, they undergo a reversible one electron reduction to the trianion. These two clusters can replace ferredoxins in a hydrogen evolving system using $\text{Clostridium pasteurianum}$ hydrogenase with dithionite as the electron donor; we believe this is the first demonstration of such biological activity.     

Charge distribution in electron transport components: cytochrome c and cytochrome c oxidase mixtures

Mixtures of cytochrome $\text{c}$ oxidase and cytochrome $\text{c}$ have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O₂. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome $\text{a}$ heme is dependent upon whether oxidation or reduction is occurring.     

Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

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 $\text{A. cylindrica}$.     

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [¹⁴C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [¹⁴C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [¹⁴C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 $\text{in vivo}$, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Chromatographic detection of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12.

Two valine-sensitive acetohydroxy acid synthase activities were separable from $\text{Escherichia}\text{coli}$ K-12 cells by virtue of their different affinities for DEAE-cellulose eluted with a KC1 gradient. These activities appeared to be independent from a valine-resistant cryptic component expressed only in $\text{ilvO}$ regulatory mutants. The properties of the first and second activity were coincident to those of extracts of $\text{ilvB}$ and $\text{ilvHI}$ mutants, respectively. These data prove that the $\text{ilvB}$ and $\text{ilvHI}$ gene products exist in the cell as physically distinct acetohydroxy acid synthase isoenzymes.     

Coordinated, coenzyme Q reversible, 2,5-dibromothymoquionine inhibition of electron transport and ATPase in Escherichia coli.

2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg⁺⁺ stimulated ATPase of $\text{E. coli}$ membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q₆, and other analogs show partial reversal by coenzyme Q₆. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg⁺⁺ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in $\text{E. coli}$ electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.     

Studies on mechanism of double hydroxylation. I. Evidence for participation of NADH-cytochrome c reductase in the reaction of benzoate 1,2-dioxygenase (benzoate hydroxylase).

Benzoate 1,2-dioxygenase system which catalyzed double hydroxylation of benzoate was obtained from $\text{Pseudomonas arvilla}$ and was shown to consist of two protein components (component A and B). Component A which was purified and was shown to be homogeneous upon sodium dodecyl sulfate disc gel electrophoresis retained high activity of NADH-cytochrome $\text{c}$ reductase. Both of benzoate 1,2-dioxygenase activity and NADH-cytochrome $\text{c}$ reductase activity were simultaneously induced by benzoate. Dichlorophenolindophenol which could serve as an electron acceptor of the NADH-cytochrome $\text{c}$ reductase inhibited the activity of benzoate 1,2-dioxygenase. These results suggest the possibility that NADH-cytochrome $\text{c}$ reductase activity is required for benzoate 1,2-dioxygenase.     

Amino acid sequence of the heme a subunit of bovine heart cytochrome oxidase and sequence homology with hemoglobin.

The amino acid sequence of the 11.6 K dalton heme $\text{a}$ subunit of bovine heart cytochrome oxidase has been completed and is presented here. The sequence investigation has established the positions in the protein of all the possible heme ligands, namely cysteine, methionine, histidine and lysine residues. However, the isolation conditions may have caused the heme $\text{a}$ to migrate from its original site or the heme is caged by peptides as pointed out in Reference 6. The sequence of the heme $\text{a}$ subunit and the β-chain of hemoglobin shows homology. It is possible that these two proteins have arisen from a common ancestor in the distant past.     

Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C., reinhardtii

An anaerobic incubation period of varying duration is required to induce hydrogenase activity in $\text{C.}\text{,}\text{reinhardtii}$. Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Reconstitution of cyanobacterial photophosphorylation by a latent Ca2+-ATPase.

Photosynthetic membranes derived from sonic extracts of the cyanobacterium $\text{Spirulina}\text{platensis}$ contain a latent Ca⁺²-ATPase which is activated by exposure to trypsin. When sonic membranes are washed with ethylenediaminetetraacetic acid, the ATPase is removed from these membranes with an accompanying loss of photophosphorylation activity. The latent ATPase activity solubilized by washing has been partially purified, and addition of the enzyme to depleted membranes restores photophosphorylation activity to levels approaching 50% of the rates observed in unwashed membranes. These data indicate that this ATPase is the coupling factor responsible for photosynthetic energy transduction in $\text{Spirulina}\text{platensis}$.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

Quinones as hydrogen carriers for a late step in anaerobic heme biosynthesis in Escherichia coli

A late step in anaerobic heme synthesis, the oxidation of protoporphyrinogen with fumarate as electron acceptor, was studied in extracts and particles of Escherichia coli mutants deficient in quinones or cytochromes. Mutants specifically deficient in menaquinone did not couple protoporphyrinogen oxidation to fumarate reduction, whereas mutants containing menaquinone but deficient in either ubiquinone or cytochromes exhibited this activity. These findings indicate that this coupled reaction is dependent upon menaquinone as hydrogen carrier but independent of ubiquinone and cytochromes. Other characteristics of this coupled reaction were also studied. The activity was located exclusively in the membrane fraction of cell-free extracts. Coproporphyrinogen III could not replace protoporphyrinogen as substrate. Methylene blue, triphenyl tetrazolium and nitrate, but not nitrite, could replace fumarate as anaerobic hydrogen acceptor. These findings have implications for the mechanism and regulation of microbial heme and chlorophyll synthesis and for the physiology of cytochrome synthesis in anaerobic microorganisms.     

The occurrence of glutamate synthase in algae.

The presence of glutamate synthase in the green algae $\text{Chlorella fusca}$ var. $\text{vacuolata}$ has been demonstrated using a whole cell assay as well as cell free extracts. The assay is complicated by the presence of glutamine (amino): α-oxoglutarate transaminase, but this enzyme can be inhibited by amino oxyacetate. The rates of glutamate synthase activity are sufficient to account for the known rates of nitrate assimilation to occur via the glutamine synthetase/glutamate synthase pathway.     

On the effect of rfe mutation on the biosynthesis of the 08 and 09 antigens of E. coli.

From phosphomannose isomerase-less mutants of $\text{E. coli}$ strains 08 and 09, $\text{rfe}{}^{\mathrm{?}}$ derivatives were constructed by recombination with a $\text{Salmonella rfe}{}^{\mathrm{?}}$ donor. In contrast to membranes from the parent $\text{E. coli}$ strains, those from the $\text{rfe}{}^{\mathrm{?}}$ recombinants did not synthesize the 08 or 09 mannan from GDP mannose $\text{in vitro}$. They could, however, be restored to biosynthetic activity with butanol extracts from the $\text{E. coli rfe}{}^{\mathrm{+}}$ bacteria. This indicated that the $\text{rfe}$ mutation affects the synthesis of a hydrophobic acceptor.