Use of 14C-carboxyl-luciferin in determining the mechanism of the firefly luciferase catalyzed reactions

The use of ¹⁴C-carboxyl-labelled luciferin as a substrate for the firefly luciferase catalyzed reaction produces ¹⁴CO₂ as a product. We have studied this reaction in the presence of ¹⁷O₂ and H¹⁸OH, using an excess of luciferin over luciferase. The initial collection of CO₂ contained close to one oxygen from ¹⁷O₂ for each molecule of ¹⁴CO₂ derived from luciferin, which is consistent with a cyclic peroxide mechanism. About half of the ¹⁴CO₂ remained bound to the enzyme and was collected after acidification of the medium. This CO₂ contained less than 0.1 of an atom of oxygen from ¹⁷O₂ for each molecule of ¹⁴CO₂ derived from luciferin. Exchange of medium CO₂-HCO₃ su? with water was not sufficiently great to account for the loss of any ¹⁷O previously incorporated. The most likely explanation appears to be a preferential exchange of oxygens of enzyme-bound CO₂ with water oxygens. Such exchange, and dilution of CO₂ from luciferin by medium CO₂, may explain previous results in which little incorporation of atmospheric oxygen was noted.     

cAMP mediated decrease in membrane phosphorylation.

Mass spectral analyses of the CO₂ liberated in the $\text{Cypridina}$ luciferin-luciferase and firefly luciferin-luciferase reactions run in the presence of ¹⁷O₂ and H₂¹⁸O show that the product is predominantly C¹⁸O¹⁶O (mass 46) and not C¹⁷O¹⁶O (mass 45). Incorporation of ¹⁸O into medium CO₂ by exchange does not account for the observed results. These experiments provide evidence that the $\text{Cypridina}$ and firefly bioluminescence reactions proceed via a linear peroxide mechanism rather than the dioxetane mechanism and suggest that a common mechanism may underly many bioluminescence reactions.     

Mechanism of oxidation of carbon monoxide by bacteria

Double label experiments were performed employing ¹³CO and either H₂¹⁸O or ¹⁸O₂ in the presence of a CO utilizing bacterium. CO₂ generated was trapped and $\text{m}\text{e}$ ratios $\text{47}\text{45}$ showed that the second oxygen atom in the oxidation of CO to CO₂ by this bacterium comes neither from O₂ nor H₂O.     

Exchange of oxygen between solvent H 2 O and the CO 2 produced in Cypridina bioluminescence

Bioluminescent oxidation of $\text{Cypridina}$ luciferin yields CO₂ besides oxyluciferin and light. The exchange of oxygen between the CO₂ and H₂O of the solvent becomes significant when less than approximately 1 μmol of luciferin is reacted in 4 ml of buffer solution, and the exchanged oxygen in CO₂ markedly increases by decreasing the amount of luciferin. Such an exchange is to be expected in any such system which produces CO₂ in aqueous solution, and must be taken into account in interpreting the results of experiments.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Hydrogen bonds in the tripeptide Pro-Leu-Gly-NH217O and 1H studies

¹⁷ONMR measurements of labeled Pro-Leu-Gly-NH₂ were carried out at different pH levels and in mixed solvents of water/acetonitrile. Complementary studies of the amide protons were carried out in acetonitrile-d₃. Only the prolyl $\text{C}\text{=}{}^{17}\text{O}$ group was sensitive to the pH level. Protonation of the amine group resulted in an upfield chemical shift of 18 ppm. The chemical shifts of each of the three oxygen sites was sensitive to the ratio water: acetonitrile. Solvent composition dependence of the chemical shift and linewidth suggests that the prolyl $\text{C}\text{=}{}^{17}\text{O}$ is involved in intramolecular hydrogen bond formation when Pro-Leu-Gly-NH₂ is dissolved in acetonitrile, while in water there is no intramolecular H bond.     

THE OXIDATION-REDUCTION POTENTIAL OF THE LUCIFERIN-OXYLUCIFERIN SYSTEM

The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate (E_o ^'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone (E_o ^'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an E_o ^'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl₂, TiCl₃, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K₃Fe(CN)₆ in presence of luciferase but without oxygen never gives luminescence.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Oxygen-18 studies on the oxidative deamination mechanism of alicyclic primary amines in rabbit liver microsomes

The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of ¹⁸O₂ or in a medium containing H₂¹⁸O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.5–336}\text{min}$) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Incorporation of 18 O into homovanillic acid of rat brain during exposure to oxygen 18 containing atmospheres

Rats were exposed to air containing ¹⁸O₂ at atmospheric pressure. $\text{In vivo}$ incorporation of ¹⁸O in brain homovanillic acid (HVA) was determined by gas chromatography-mass spectrometry. One ¹⁸O atom was incorporated into each molecule of HVA indicating that tyrosine is the predominant precursor of brain dopamine and that the oxygen in the 3-position is of atmospheric origin. Intraperitoneal administration of ¹⁸O-enriched water did not alter the ¹⁸O content of brain HVA Mass fragmentography (2) was used to measure the increase in ¹⁸O and the decrease in ¹⁶O in HVA from rat brain over several hours of exposure to an ¹⁸O enriched atmosphere. These experiments demonstrate the possibility to pulse label brain dopamine and its metabolites by $\text{in vivo}$ inhalation of stable oxygen isotopes. The procedure should be useful for quantitative determinations of the turnover of brain dopamine in animals and man.     

Energy coupling in lysolecithin-treated submitochondrial particles.

Mitochondria isolated from mung bean hypocotyls, possessing a significant level of cyanide and antimycin A — resistant respiration $\text{via}$ an alternate pathway, were assayed for hydrogen peroxide production by yeast cytochrome $\text{c}$ peroxidase compound II formation. Rates of antimycin A — insensitive hydrogen peroxide production of 0.7–3 nmol/mg/min were observed which were too low to account for the observed oxygen consumption $\text{via}$ the alternate pathway. However, further investigations revealed the presence of significant levels of catalase, peroxidase and hydrogen donor to peroxidase, even in gradient purified mitochondria and these could easily utilize any hydrogen peroxide produced by the alternate pathway. Similar experiments performed upon submitochondrial particles demonstrated a rate of H₂O₂ production which could easily account for the net electron flux through the alternate pathway. From these results, we postulate that the alternate pathway reduces oxygen only partially to hydrogen peroxide, and that the peroxidase and catalase activities of the mitochondria prevent its accumulation.     

H/2H isotope effect in redox reactions of cytochrome c

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O₂^? and CO₂^? was determined in H₂O and in ²H₂O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H₂O and ²H₂O. An isotope effect with , depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

The effect of oxygen on 14CO2 fixation in mesophyll cells isolated from Digitaria sanguinalis (L.) Scop. leaves

Mesophyll cells were isolated from fully-expanded leaves of $\text{Digitaria sanguinalis}$ (L.) Scop. by a combined maceration-filtration technique. In the presence of pyruvate, photosynthetic ¹⁴CO₂ uptake in the isolated cells was not inhibited by atomospheric levels of oxygen. In contrast, superatmospheric levels of oxygen substantially inhibited the light-dependent fixation of ¹⁴CO₂. These oxygen effects are similar to those observed with intact C4 leaves and suggest that the lack of inhibition of C4 photosynthesis by atmospheric levels of oxygen results from the relative oxygen-insensitivity of the phosphopyruvate carboxylase-CO₂ pump in the mesophyll.     

Novel heme: O2 bonding of possible relevance to oxygen utilizing heme and other proteins

Solid dipyridine hemes which are unreactive toward oxygen lose both pyridine ligands upon heating under vacuum to give a solid which takes up O₂, reversibly, one O₂ per heme. Replacement of ¹⁶O₂ by ¹⁸O₂ reduces only infrared bands near 1660 and 1590 cm^?1, frequencies near the vibrational band for gaseous O₂. No FeO bands are detected. EPR spectra reveal a free radical and ferric iron; Mössbauer, NMR and infrared spectra support an iron(III) oxidation state. Limited molecular weight data indicate a dimer. Possibly two dioxygen molecules are held sandwich fashion between two porphyrins via donor-acceptor interactions, which are facilitated by electron transfer from iron(II) into the porphyrin forming a π-anion. Such O₂ bonding is not found in oxy Hb and Mb or in oxyhemerythrin but may occur with cytochrome $\text{c}$ oxidase and other oxygen utilizing (or producing) heme and other proteins.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Involvement of singlet oxygen in the fungal degradation of lignin

The possible involvement of singlet oxygen (¹O₂) in the degradation of lignin by $\text{Phanerochaete}\text{chrysosporium}$ was examined. Ligninolytic cultures and photochemically generated ¹O₂ gave the same oxidation products from the lignin substructure model compound 1,2-bis(3-methoxy-4-alkoxyphenyl)propan-1,3-diol. Fluorescence and near UV absorbance of the specific ¹O₂ trapping agent anthracene-9,10-bisethanesulfonic acid (AES) disappeared in ligninolytic cultures, indicating that ¹O₂ was produced. AES strongly inhibited oxidation of ¹⁴C-lignin, but not ¹⁴C-glucose, to ¹⁴CO₂ in cultures, and also strongly suppressed oxidation of the model compound. These results indicate the ¹O₂ plays an integral role in lignin biodegradation.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.