Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediaesp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.     

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.     

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Effect of pH on the spectrum of cytochrome c oxidase hydrogen peroxide complex

Hydrogen peroxide binding to ferric cytochrome c oxidase in proteoliposomes brings about a red-shift of the enzyme Soret band and increased absorption in the visible range with two prominent peaks at approx. 570 and 607 nm. The molar absorptivity of the H2O2-induced difference spectrum is virtually pH-independent in the Soret band and at 570 nm, whereas the peak at 607 nm increases approx. 3-fold upon alkalinization in a narrow pH range 6.0-7.2, the effect being reversible. The pH profile of this transition indicates ionization of two acid-base groups with close pK values of 6.7. The lineshape of the peroxide compound difference spectrum is found to respond to pH changes inside the proteoliposomes. It is suggested that peroxide-complexed enzyme can undergo a pH-dependent transition to a form with increased extinction at 605-607 nm, possibly corresponding to the 420 nm (or 'pulsed') conformer of the ferric cytochrome oxidase formed as an early product of the enzyme oxidation. Accordingly, relaxation of the '420 nm' form to the resting state would be linked to an uptake of two protons from the M-aqueous phase. This protolytic reaction might be a partial step of the cytochrome oxidase proton pumping mechanism or it could serve to regulate interconversion between the active 'pulsed' and less active 'resting' states of the enzyme in the membrane.     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.     

Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.     

Cytochrome c oxidase as a calcium binding protein. Studies on the role of a conserved aspartate in helices XI-XII cytoplasmic loop in cation binding

The aa(3)-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established. D477A mutation in subunit I of P. denitrificans oxidase converts the cation-binding site to a mitochondrial-type form that binds reversibly calcium and sodium ions [Pfitzner, U., Kirichenko, A., et al. (1999) FEBS Lett. 456, 365-369]. We have studied reversible cation binding with P. denitrificans D477A oxidase and compared it with that in bovine enzyme. In bovine oxidase, one Ca(2+) competes with two Na(+) for the binding, indicating the presence of two Na(+)-binding sites in the enzyme, Na(+)((1)) and Na(+)((2)). In contrast, the D477A mutant of COX from P. denitrificans reveals competition of Ca(2+) (K(d) = 1 microM) with only one sodium ion (K(d) = 4 mM). The second binding site for Na(+) in bovine oxidase is proposed to involve D442, homologous to D477 in P. denitrificans oxidase. A putative place for Na(+)((2)) in subunit I of bovine oxidase has been found with the aid of structure modeling located 7.4 A from the bound Na(+)((1)) . Na(+)((2)) interacts with a cluster of residues forming an exit part of the so-called H-proton channel, including D51 and S441.     

Zinc ions as cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase inhibitors: two sites of action

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50–90%,depending on preparation) with an effective K _i of 10 µM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes,and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme:full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 µM. The rate of zinc inhibitory action in the slow phase is proportional to Zn²⁺ concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F O step corresponding to transfer of the 4th electron in the catalytic cycle).Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 160–170.Original Russian Text Copyright © 2005 by Kuznetsova, Azarkina, Vygodina, Siletsky, Konstantinov.This revised version was published online in April 2005 with corrections to the post codes.