Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

Effects of Salicylate on Virus-Infected Tobacco Plants

Salicylate watered onto the soil of tobacco plants in pots reduced the antigen accumulation and local lesion growth of tobacco necrosis virus mechanically inoculated on the leaves. It also retarded the growth of the necrotic centres of lesions and, in parallel, inhibited ethylene production from infected leaves. However, the therapeutic index of salicylate was very small and the chemical had to be applied in advance of, or at the same time as virus inoculation to give good levels of resistance. The number of lesions and their rate of appearance were not affected by salicylate. In addition, it did not induce resistance against multiplication, systemic spread or symptom severity in tobacco plants inoculated with a necrotic strain of potato virus Y. These findings suggest that salicylate is not likely to prove useful as polyvalent chemotherapeutic agent.     

Blood amino acids in chronic HBsAg positive liver disease

Typical changes in blood aminoacid concentrations have been described in patients with severe liver disease. In this study we measured the serum amino acid levels, by Beckman Aminoacid Analyzer, in 11 healthy subjects and 24 HBsAg-positive patients with biopsy-proven liver disease (4 CPH, 10 CAH, 10 cirrhosis). A significant decrease in total aminoacids was observed in CAH and cirrhosis groups (-24% and -22% respectively). The three branched chain aminoacids (BCAA = val + leu + isoleu) were reduced by 24% (P less than 0.002) and 37% (P less than 0.001) in the CAH and cirrhosis groups respectively. Tyrosine was the only of the aromatic aminoacids (AAA) to increase in cirrhotics (+ 34%, P less than 0.02). The molar ratio BCAA/AAA was 3.6 in controls, 3.8 in CPH, 3.1 in CAH (P less than 0.025) and 1.9 in cirrhosis (P less than 0.001). A linear correlation was found between molar ratio BCAA/AAA and serum albumin in all patients (P less than 0.001). These results document the presence of specific quantitative changes in serum aminoacids of HBsAg positive patients, which appear related to severity of liver disease and comparable to the alterations described in non viral chronic liver disease.     

Possible role of 06-methylguanine-DNA-transferase in the response of patients with primary tumor of the brain to chemotherapy using chloro-ethyl-nitroso-urea: results of a current study

We have started a study to measure the MT activity in surgical specimens from high grade human malignant gliomas, with the dual aim to (i), know whether lack of activity can be demonstrated in these tumors, and (ii), relate the measured levels of MT to the histology of the tumors and to the response of patients to chemotherapy with 1-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosourea (CCNU). To date, 12 Gliomas have been assayed. In 11 tumors, MT activities ranging from 30 to 150 fmoles/mg protein have been measured. The only negative specimen derived from a patient who had received radiotherapy before surgery. At the present stage of the study, therefore, we have no unequivocal evidence for the existence of MT-deficient Gliomas.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.