Organic Cofactor Research Articles

Domains in bovine serum amine oxidase

Analysis of the thermal unfolding of bovine serum amine oxidase by differential scanning calorimetry reveals for the dimeric protein a four domain structure consisting of two sets of domains. Each set contains two domains of similar size. The two smaller domains, in contrast with the larger ones, greatly differ in thermostability. Removal of copper changes the calorimetric pattern dramatically. The findings confirm that the metal cofactor plays a structural role. Since the enzyme contains two copper atoms and only one titratable carbonyl group, the calorimetric pattern suggests that the difference in thermostability of the two small domains might be due to the presence of a single organic cofactor.

The haemocyanin of the tarantula Lasiodora erythrocythara—the influence of CO 2, organic cofactors and temperature on oxygen affinity

1. 1. Whole blood oxygen equilibrium curves for the haemocyanin of the Honduran tarantula Lasiodora erythrocythara have for the first time been investigated in vitro. 2. 2. Oxygen affinity was pH dependent with a mean Bohr coefficient of −0.71 ± 0.06 between pH 7.3 and 8.1. At 25°C and pH 7.6 the P 50 value was 23 Torr. 3. 3. The fixed acid Bohr effect was not significantly different from the CO 2 Bohr effect indicating the lack of a specific effect of CO 2 on oxygen affinity. 4. 4. Dialysis had no measurable effect on haemocyanin oxygen affinity, indicating the absence of unidentified dialysable factors. The haemocyanin was also insensitive to changes in the concentration of d and l lactate, succinate, guanosine and urate. 5. 5. Temperature was found to have little influence on oxygen affinity between 20 and 25°C illustrated by the low Δ H value of −5.6 kJ mol −1. At the temperature extremes of 15 and 30°C Δ H values increased to −29kJmol −1. 6. 6. Cooperativity, expressed as n 50, was both temperature and pH dependent.

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.

On the nature of the organic cofactor of pig plasma benzylamine oxidase

On the nature of the organic cofactor of pig plasma benzylamine oxidase

Reactions of bovine serum amine oxidase with NN-diethyldithiocarbamate. Selective removal of one copper ion.

NN-Diethyldithiocarbamate (DDC) was able to bind, at 1.0 mM concentration, only about 50% the Cu(II) ions of bovine plasma amine oxidase. Under reducing conditions, this Cu(II) was removed with inactivation of the enzyme. Up to 90% activity could be recovered by treatment with excess Cu(II). The organic cofactor, sensitive to carbonyl reagents, was reduced in the half-Cu-depleted protein and no longer bound phenylhydrazine. The fully reacted protein, in the presence of 10 mM-DDC, lost 50% Cu(II) upon storage at -20 degrees C, but in this case the residual Cu(II) was in the DDC-bound form and the cofactor was in the oxidized state, as it could still bind phenylhydrazine. In the presence of DDC, the rate of reaction with phenylhydrazine was always low, even at 50% DDC saturation, and all derivatives showed identical modifications of the optical and e.p.r. spectra with respect to the phenylhydrazone of the native protein. It is concluded that the two Cu(II) ions are not equivalent, that removal of a single Cu(II) is sufficient to inhibit the re-oxidation of the organic cofactor, and that both Cu(II) ions are in some way involved in the reaction with phenylhydrazine. After reaction with DDC, the optical and e.p.r. spectra of 63Cu(II)-amine oxidase and of 63Cu(II)-carbonic anhydrase [Morpurgo, Desideri, Rigo, Viglino & Rotilio (1983) Biochim. Biophys. Acta 746, 168-175] are very similar and show distorted equatorial co-ordination to Cu(II) of two sulphur atoms and two magnetically equivalent nitrogen atoms.

Tyrosine radicals are involved in the photosynthetic oxygen-evolving system.

In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.

The organic cofactor in plasma amine oxidase: evidence for pyrroloquinoline quinone and against pyridoxal phosphate.

Plasma amine oxidases (EC 1.4.3.6) are classified as containing the organic cofactor pyridoxal phosphate. Biochemical and bioassays on the pig plasma amine oxidase fail to reveal the presence of pyridoxal phosphate and 31P n.m.r. evidence is also inconsistent with pyridoxal phosphate in the enzyme. Resonance Raman spectral studies on phenylhydrazone derivatives of the pig and bovine plasma enzymes have been carried out and comparisons made with the corresponding derivatives of pyridoxal phosphate and pyrroloquinoline quinone (PQQ). The resonance Raman evidence indicates that the cofactor in both plasma amine oxidases is PQQ or a closely related species and not pyridoxal phosphate. The results substantiate earlier reports concerning the identity of the organic cofactor.

Evidence for methoxatin (pyrroloquinolinequinone) as the cofactor in bovine plasma amine oxidase from resonance Raman spectroscopy.

Resonance Raman spectra of the 2,4-dinitrophenylhydrazine derivatives of bovine plasma amine oxidase [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] have been measured. Detailed comparisons to the spectra of the corresponding derivatives of methoxatin (pyrroloquinolinequinone), pyridoxal, and other aldehydes and diones provide further evidence that covalently bound methoxatin or a closely similar derivative is the organic cofactor in copper-containing amine oxidases.

Electron nuclear double-resonance (ENDOR) spectroscopy of amine oxidase from pig plasma

Electron nuclear double-resonance ('ENDOR') spectroscopic studies on pig plasma amine oxidase have been carried out at 15 K. Deuterium-exchange studies show the presence of two sets of exchangeable protons, probably from two water molecules; from the magnitude of their hyperfine couplings, one is assigned to be equatorially, and the other axially, co-ordinated. Only one 14N hyperfine coupling is observed, suggesting that the bonding of all amino acid (histidine) or organic cofactor ligands is similar. Upon addition of azide, a further hyperfine coupling to nitrogen is observed which is smaller than that observed for the native enzyme; the hyperfine couplings to the remaining nitrogens are slightly altered.

Inactivation of beef plasma amine oxidase by sulfide.

Sulfide irreversibly inactivates beef plasma amine oxidase in a time-dependent reaction. Mercaptoacetic acid and 2-mercaptoethanol do not inactivate the enzyme. The sulfide complex displayed an intense absorption band at 360 nm (epsilon = 6000 M-1 cm-1, per mol of copper) that is assigned as sigma S leads to Cu(II) ligand to metal charge-transfer transition. However, this band slowly decreased in intensity; the final spectrum resembles the spectrum of the dithionite-reduced enzyme. Bleaching at approximately 450-500 nm specifically indicates that the organic cofactor is reduced. EPR parameters for the sulfide complex differ significantly from those observed for the native amine oxidase. Superhyperfine structure, attributable to coordinated nitrogens, is clearly evident. Time-dependent reduction of Cu(II) that parallels the kinetics and absorbance changes was also observed by EPR. The amine oxidaseazide complex was inactivated by sulfide at a considerably slower rate than the resting enzyme. Since azide is known to coordinate to Cu(II) in beef plasma amine oxidase, the data strongly suggest that enzyme-bound copper is the site of action for inhibition by sulfide.

Inhibition of copper-containing amine oxidases by Cu(II) complexes and anions

As part of an effort to elucidate the mechanism of O 2 reduction by amine oxidases and the role of copper in the overall reaction, inhibition by Cu(II) complexes and anions has been investigated. Copper(II) complexes of 1,10-phenanthroline and 2,2'-bipyridine strongly inhibited both beef plasma and pig kidney amine oxidase. Lysine, tyrosine and salicylate complexes, as well as aqueous Cu 2+, inhibited the plasma but not the kidney amine oxidase. A variety of control experiments indicate that the most plausible mechanism for the inhibition is O 2 − dismutation (intercepting a reaction intermediate) within an active site that is somewhat inaccessible to external reagents. Reactivity differences between pig kidney and beef plasma amine oxidase can also be rationalized on the basis of active site accessibility. Azide and thiocyanate are effective inhibitors of both amine oxidases. The data suggest that inhibition results from coordination to Cu(II) by N 3 − and SCN −. Ligand-to-metal charge-transfer transitions are prominent in the absorption spectra of the N 3 − (λ max $ ̃ 400 nm, ϵ ~ 3300–6300 M −1 cm −1) and SCN − (λ max ~ 370 nm, ϵ ~ 3000 M −1 cm −1) complexes with both amine oxidases. The position and intensity of these bands establish that the anions are equatorially coordinated in a tetragonal Cu(II) complex. Substrate reduction alters the anion binding properties of Cu(II) in beef plasma amine oxidase. The results are consistent with a mechanistic role for copper and indicate that the copper site is sensitive to reactions at the organic cofactor.

Iron and manganese superoxide dismutases

Fe and Mn SDs are enigmatic metalloproteins for several reasons: They are found in strict anaerobes as well as aerobes. In some cases the Fe and Mn ions appear to be interchangeable while in others they are not. In some organisms the Mn-protein is strongly induced by O 2 while in others the Fe-protein is induced. The two distinct types of proteins are structurally related as judged from published amino acid sequences. The Fe-protein appears to have a non-covalently associated organic co-factor bound near the iron (see below). Recently, the three dimensional structures of FeSD from E. coli and Ps. ovalis have been elucidated in the laboratories of M. Ludwig and G. Petsko. The FeSD molecule is composed of a dimer of identical subunits related within the crystal by a dyad axis. The distance between the Fe atoms is 18 Å and each is close to the subunit interface. Access to the Fe appears to be from a region near the interface. Each monomer has two distinct structural domains, connected by a single strand and the Fe is bound at the interface of the two domains receiving two ligands from each. The Fe is surrounded by four ligands from the protein and probably a water molecule. The geometry, at 3 Å resolution has the appearance of a flattened pyramid. According to present interpretations of the electron density map the four protein ligands are His-26, residue 69, and residues 148 and 152. The spectral properties of the protein would appear to exclude tyrosine as a ligand to the Fe. A region of unconnected electron density is found in the region of the molecule separating the two domains and about 6 Å from the Fe. This is a dominant feature in FeSD from both sources and may represent an organic co-factor. To date we have not isolated and identified this material. We are involved in a program to correlate structural functional properties of the FeSD from E. coli. The presentation will be concerned with redox probes of the Fe, steady state and transient kinetic studies of superoxide dismutation, and relations between anion inhibition and binding to the Fe. If time allows a comparison will be made between the mechanism of FeSD action and the weak superoxide dismutase activity of Fe-EDTA.

Cryoenzymology and spectrophotometry of pea seedling diamine oxidase.

Diamine oxidase follows bi-ter ping-pong kinetics, with an intermediate, "reduced" free-enzyme form being generated after the anaerobic conversion of amine to aldehyde. Visible spectra of diamine oxidase reacting at subzero temperatures provide evidence that this intermediate enzyme form is obtained via several other intermediates and that the environment of the Cu(II) changes dramatically during the course of the reaction [even though it is not reduced to Cu(I) during the catalytic cycle]. The spectrum of this form of diamine oxidase, which is obtained 0.5--2 h after the addition of amine at -5 to -15 degrees C, is independent of substrate, is identical with that obtained by anaerobic addition of substrate at room temperature, and provides evidence for a direct interaction of Cu(II) with the organic cofactor of the enzyme. This interaction is apparently charge transfer in nature. Upon removal of Cu(II) from the native enzyme, one obtains spectral evidence that the organic cofactor is still present. However, removal of the Cu(II) from the reduced (intermediate) enzyme form yields a featureless enzyme spectrum and a Cu(II)--chelate complex which contains a new ligand, which is presumably the second prosthetic group.

APPLICATIONS OF CALCIUM‐SENSITIVE PHOTOPROTEINS IN EXPERIMENTAL BIOLOGY

Abstract—Calcium‐sensitive photoproteins are “precharged bioluminescent proteins that are triggered to emit light by binding Ca2+ or certain other inorganic ions. Neither molecular oxygen nor any organic cofactor is required. The first such protein to be described was aequorin, and for various reasons that has been the one most widely studied. Photoproteins have been used as Ca2+‐indicators both in vitro and in living cells. Their chief advantages for this are (1) ease of signal detection, (2) high sensitivity, (3) relative specificity for Ca2+, and (4) lack of toxicity. Difficulties in the experimental use of the photoproteins stem from (1) their one‐time reactivity, (2) their large molecular size, (3) their scarcity, (4) the influence of experimental conditions on the sensitivity to Ca2+, (5) the nonlinearity of the relation between [Ca2+] and light intensity, and (6) the limited speed with which light intensity follows sudden changes in [Ca2+]. Photoproteins have now been used as intracellular calcium indicators in more than two dozen types of cells, and experience with the method is rapidly growing. They are also useful in the determination of calcium binding constants for other substances in vitro, and as models for studies of receptor‐ligand interactions in general.

Nutrition of Myxococcus xanthus, a fruiting myxobacterium

The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.

Anion modulation of the negative Bohr effect of haemoglobin from a primitive amphibian

THE Congo eel (Amphiuma means) is a primitive salamander that respires through its lungs, gills and integument. It lives in swamps, ditches and rice fields in the south-eastern USA. When pools and swamps become stagnant and water levels drop, Amphiuma will burrow and hibernate in the mud. In this letter we report that when organic phosphate cofactors are low or absent, as might be true during hibernation, the haemoglobin of this amphibian has unusual properties which may help the animal to extract oxygen from a low pH environment. The molecular adaptation exhibited by Amphiuma haemoglobin is a pH dependence of oxygen binding that is reversed relative to that of most haemoglobins. Low pH brings about a large increase in the oxygen affinity of stripped Amphiuma haemoglobin. This negative Bohr effect indicates that protons are bound as oxygen is bound. This is in contrast to the release of protons that usually accompanies oxygenation. New Bohr groups must be present in this amphibian haemoglobin, or the normal Bohr groups must be drastically modified. From a physiological viewpoint it is of interest that the Bohr effect in vitro is subject to regulation by organic phosphate cofactors.

Riboflavin Synthetase from Yeast: PROPERTIES OF COMPLEXES OF THE ENZYME WITH LUMAZINE DERIVATIVES AND RIBOFLAVIN

Abstract A general mechanism for the riboflavin synthetase reaction involving the addition of 6,7-dimethyl-8-ribityllumazine at two sites on the enzyme has been proposed. One site binds the substrate in such a way that it functions as donor of the 4-carbon moiety transferred in the riboflavin synthetase reaction, while the other binds the lumazine that serves as acceptor of the 4-carbon fragment. The rate equation derived from such a reaction mechanism contains a term second order with respect to the concentration of 6,7-dimethyl-8-ribityllumazine, indicating a possible nonlinear relationship between the reciprocals of velocity and 6,7-dimethyl-8-ribityllumazine concentration. However, zero to first order kinetics were observed over a range of substrate concentration from 2 x 10-4 m to 6 x 10-7 m (compared to 1.0 x 10-5 m for the Michaelis constant) suggesting that binding of the 2 molecules of the lumazine to the enzyme occurs with widely different affinities. Riboflavin synthetase also catalyzes the conversion of 6,7-dimethyl-8-(5'-deoxyribityl)lumazine to 5'-deoxyriboflavin at a slow rate. However, rapid formation of riboflavin-14C (but not 5'-deoxyriboflavin-14C) occurs in the presence of a mixture of equimolar amounts of 6,7-dimethyl-8-ribityllumazine and 6,7-dimethyl-14C-8-(5'-deoxyribityl)lumazine. This implies that 6,7-dimethyl-8-(5'-deoxyribityl)lumazine is an efficient donor of the 4-carbon fragment involved in the formation of the o-xylene portion of riboflavin, but functions poorly as an acceptor of the 4-carbon moiety. Riboflavin synthetase, purified 2000-fold from an extract of bakers' yeast, forms isolatable complexes with substrate (6,7-dimethyl-8-ribityllumazine or 6,7-dimethyl-8-(5'-deoxyribityl)lumazine), riboflavin, and certain inhibitory substrate analogues (e.g. 6-methyl-7-hydroxy-8-ribityllumazine or 6,7-dimethyl-8-d-xylityllumazine). Only those substances are bound which have a kinetic effect on the enzyme; e.g. 6,7-dimethyl-8-l-xylityllumazine does not inhibit activity and is not bound. The complexes contain riboflavin, substrate, or analogues of 6,7-dimethyl-8-ribityllumazine, respectively, in equivalent molar amounts. The binding is not due to covalent bonding since riboflavin can be removed from the protein with charcoal; the treated enzyme retains activity. Furthermore, a tightly bound compound can be rapidly and completely displaced by an excess of the same or another substance with which the enzyme can combine. Thus, displacement has been shown of bound riboflavin by the inhibitory substrate analogue 6-methyl-7-hydroxy-8-ribityllumazine and of bound 6,7-dimethyl-14C-8-ribityllumazine by unlabeled 6,7-dimethyl-8-ribityllumazine. The substrates are probably bound in the complex at the site which leads to donation of the 4-carbon moiety since reaction of equivalent amounts of enzyme-6,7-dimethyl-14C-8-(5'-deoxyribityl)lumazine with 6,7-dimethyl-8-ribityllumazine yields riboflavin-14C only, and not 5'-deoxyriboflavin-14C. Titration of enzyme-6,7-dimethyl-8-ribityllumazine complex with free 6,7-dimethyl-8-ribityllumazine resulted in further polarization of fluorescence. It was calculated that maximal polarization of fluorescence occurred upon the addition of 1 additional eq of substrate to the complex. These results suggest the occurrence of a second substrate-binding site on the enzyme which is involved in the acceptance of the 4-carbon moiety. Attempts to identify an intermediate of the riboflavin synthetase reaction were unsuccessful. No evidence could be obtained that the enzyme requires an organic cofactor for activity.