N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant.

Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552,+0.19 V, is markedly less than that of horse heart cytochrome c. (2) ?H_ox³ of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) ?H_ox³ and ?S_red³ of cytoochrome c-552 are more negative than those of horse heart cytochrome c. (4) k_red of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Evidence that enzymatic conversion of N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific inhibitor of endopeptidase 24.15, to N-[1 (R,S)-carboxy-3-phenylpropyl]-Ala-Ala is necessary for inhibition of angiotensin converting enzyme

N-[1 (R,S)-Carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) is a potent, substrate-related, specific inhibitor of endopeptidase 24.15, an enzyme involved in the metabolism of bioactive peptides including bradykinin, neurotensin, and proenkephalin, and prodynorphin-derived enkephalin precursors. The observation that this inhibitor causes a pronounced decrease in blood pressure after intravenous infusion into normotensive rats posed the question of the mechanism of this hypotensive response. It was suggested previously that cFP-AAF-pAB is an inhibitor of angiotensin converting enzyme (ACE) and that this function can account for the hypotensive response to the inhibitor. We present here evidence that cFP-AAF-pAB has no intrinsic ACE-inhibitory activity. The previously observed inhibition is shown to be dependent on cleavage of the Ala-Phe bond in the inhibitor by endopeptidase 24.11 (enkephalinase, EC 3.4.24.11), a contaminant of some ACE preparations.     

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c

Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80–heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80–heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

1-Acyl-2-[N-(2,4-dinitrophenyl)aminopropionyl]-sn-glycero-3-phosphocholine as a chromogenic substrate for phospholipase A2 assay

We have developed a spectrophotometric assay for phospholipase A₂ activity using 2,4-dinitrophenyl-labeled phosphatidylcholine as substrate. The assay allows quite simple quantification of phospholipase A₂ activity by measuring the absorbance of the aqueous phase after extraction of the reaction mixture and requires neither chromatographic separation of the reaction products nor the addition of auxiliary coloring reagents.     

Biochemical and biophysical studies on cytochrome c oxidase. XIX. An EPR study of the photodissociation of carboxycytochrome c oxidase in the presence of azide

1. The photodissociation reaction of the cytochrome c oxidase-CO compound in the presence of azide was studied by EPR at 15°K. Addition of CO in the dark to cytochrome c oxidase, partially reduced (2 electrons per 4 metal ions) in the presence of azide brings about a decrease in intensity of the azide-induced low-spin heme signal at g = 2.9, 2.2 and 1.67 and an increase in intensity of both the low-spin heme signal at g = 3 and the copper signal at g = 2. Subsequent illumination with white light at room temperature of this sample causes an enhancement of the azide-induced signal at g = 2.9, and a decrease in intensity of both signals at g = 3 and g = 2. It is shown that these changes in the EPR spectrum are reversible.

2. These results demonstrate that upon photodissociation, CO is replaced by azide wheras upon incubation in the dark CO expels azide from its binding site in cytochrome c oxidase.

3. Concomitantly with the binding of CO and dissociation of the azide molecule, and vice versa, electron redistributions occur as inferred from the changes in the intensity of the copper signal at g = 2.

4. The results are explained in a model of cytochrome c oxidase with either a common binding site (cytochrome a₃)^* for CO and azide or in a model with anti-cooperative interaction between two different sites of binding.

5. Similar types of experiments with cyanide instead of azide show that cyanide is more firmly bound to partially reduced cytochrome c oxidase than CO and azide. The affinity of ligands for partially reduced enzyme decreases in the sequence: cyanide, CO (dark), azide and CO (illuminated).     

Kinetic studies on 1:1 electron transfer reactions involving blue copper proteins : 8. Reactions of plastocyanin and azurin with cytochrome c and high potential iron-sulfur protein

Rate constants determined by the stopped-flow method for four protein-protein reactions at 25°C, pH's in the range 5.8–7.5. I = 0.10 M (NaCI), are as follows: cytochrome c(II) with plastocyanin, PCu(II). 1.5 × 10⁶ M⁻¹ sec⁻¹, pH 7.6; high-potential iron-sulfur protein (Hipip) with PCu(II), 3.7 × 10⁵ M⁻¹sec⁻¹. pH 5.8; cytochrome c(II) with azurin, ACu(ll). 6.4 × 10³ M⁻¹sec⁻¹, pH 6.1; Hipip with ACu(II), 2.2 × 10⁵ M⁻¹sec⁻¹, pH 5.8. Activation parameters have been determined for all four reactions; they indicate higher enthalpy requirements and less negative entropy requirements for the PCu(II) as opposed to ACu(II) reactions. Equilibrium constants K for association prior to electron transfer are < 150 M⁻¹ for the cytochrome c(II) reduction of PCu(II) (estimated charges 8 + and 9-,respectively), and < 300 M⁻¹ for the other reactions, indicating no favorable interactions. Rate constants have been analyzed in terms of the simple Marcus theory, which has previously given an excellent fit to thirteen protein-protein reactions considered by Wherland and Pecht. No similar correlation exists in the present studies, and calculated rate constants differ by orders of magnitude from experimentally determined values.     

Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea

Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine. Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO. The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin [Arciero, D.M. et al. (1993) Biochemistry 32, 9370–9378]. The recent determination of the gene sequence for cytochrome P460 [Bergmann, D.J. and Hooper, A.B. (1994) FEBS Lett. 353, 324–326] indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin.Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pK_a values for lysine and tyrosine.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

The induction of sister-chromatid exchanges in Chinese hamster ovary cells by K-region epoxides and some dihydrodiols derived from benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene

Experiments were performed to investigate the effects of 3 polycyclic aromatic hydrocarbons, benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene and K-regio epoxides and some of their related dihydrodiols on the chromosomes of Chinese hamster ovary cells in vitro. Of the 3 hydrocarbons only benz[a]anthracene showed any activity in inducing sister-chromatid exchanges. The K-region epoxide and the 3,4-dihydrodiol have been found to be more active than the corresponding K-region or the other non K-region dihydrodiols derived from benz[a]anthracene. Athough dibenz[a,c]anthracene was almost inactive, the K-region 5,6-epoxide and all 3 possible dihydrodiols, the 1,2-, 3,4- and 10,11-diols were active in inducing increased numbers of sister-chromatid exchanges in the chromosomes of these cells. The 3,4-dihydrodiol of dibenz[a,h]anthrecene was also active in inducing sister-chromatid exchanges whereas the 1,2- and 5,6-dihydrodiols were only weakly active. This study provides some support for the suggestiion that the activation of these 3 hydrocarbons proceeds by the metabolic conversion of non K-region dihydrodiols into vicinal diol-epoxides.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary The mutant T44() of Escherichia coli K12, grown in the presence of adenine, develops an increased tolerance to streptomycin. In cultures grown on streptomycin, the ts character (tif) may temporarily be suppressed but, on further transfer, both the temperature-sensitive phenotype and streptomycin tolerance disappear. In a cell-free system, the relative efficiency of translation of MS2 and poly U messenger RNAs was, respectively, 75 and 50% lower in extracts from cultures grown at 37° with adenine than in extracts from 30° cultures. Similar results were obtained when adenine was added in vitro to an extract from a culture grown at 37° in the absence of adenine, using MS2 RNA as messenger. Moreover, the 37° extracts showed a much lower misincorporation of isoleucine into polyphenylalanine in the poly U system. In addition, the Mg⁺⁺ concentration required for optimal translational activity was higher for the 37° than for the 30° extracts. Extracts from a culture grown in L medium at 37° or from a tif ^-/F tif ⁺ merodiploid grown at 37° with adenine behaved similarly to that from the 30° culture when poly U was used as messenger RNA. It is suggested that the tif ⁺ gene product may play a regulatory role in ribosomal function and the pleiotropic nature of the tif-1 mutation could be due to impairment of translational activity augmented by elevated temperature or by adenine.     

NMR studies of [1-2H]glucose metabolism in Zymomonas mobilis

In complementary experiments the metabolism of [1-2H]glucose in H2O and of unlabelled glucose in 2H2O by Zymomonas mobilis was examined. The utilization of [1-2H]glucose by Z. mobilis was monitored by high-resolution 2H NMR. The deuterium-labelling pattern and stereochemistry of the ethanols produced from the metabolism of [1-2H]glucose and unlabelled glucose in 2H2O were determined by a combination of 13C and 1H NMR and selective enzyme action. The labelling patterns were explained in terms of enzyme mechanisms and stereospecificity, and metabolite enolization.