Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase

Determining the way in which deleterious mutations interact in their effects on fitness is crucial to numerous areas in population genetics and evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last (synergistic epistasis), then the evolution of sex and recombination may be favored to facilitate the elimination of deleterious mutations. However, there is a severe shortage of relevant data. Three relatively simple experimental methods to test for epistasis between deleterious mutations in haploid species have recently been proposed. These methods involve crossing individuals and examining the mean and/or skew in log fitness of the offspring and parents. The main aim of this paper is to formalize these methods, and determine the most effective way in which tests for epistasis could be carried out. We show that only one of these methods is likely to give useful results: crossing individuals that have very different numbers of deleterious mutations, and comparing the mean log fitness of the parents with that of their offspring. We also reconsider experimental data collected on Chlamydomonas moewussi using two of the three methods. Finally, we suggest how the test could be applied to diploid species.     

Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by mutation of yme1

We have confirmed by spectral analysis that cytochrome oxidase is not present in strains of the yeast Saccharomyces cerevisiae having a primary deficiency in cytochrome c, and we have demonstrated by immunological procedures that such strains lack the mitochondrially encoded subunits I, II, and III of cytochrome oxidase. Furthermore, pulse-chase experiments demonstrated that subunit II is rapidly degraded in vivo. This degradation can be at least partially suppressed by disruption of the nuclear gene YME1, which encodes a putative ATP-Zn(2+)-dependent protease. We suggest that the cytochrome oxidase subunits are not properly assembled in the absence of cytochrome c, and that Yme1 and possibly other proteases degrade the unassembled mitochondrial-encoded subunits of cytochrome oxidase.     

Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata

The midpoint potentials of the mitochondrial respiratory chain cytochromes of the protozoan Crithidia fasciculata at pH 7.2, Em7.2, show great similarity to those measured in higher organisms. Values of Em7.2 for cytochromes a and a3 are +165 and +340 mV. Both c cytochromes have Em7.2 = +230 mV. There are two b cytochromes with the same spectral characteristics with Em7.2 = -20 and -135 mV. These values are compatible with two sites of energy conservation for oxidative phosphorylation in these mitochondria. All cytochrome components show potentiometric titrations with n = 1. There is a fluorescent flavoprotein in these mitochondria with Em7.2 = -40 mV and n =2, whose function is not known.     

Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.     

The subunit location of magnesium in cytochrome c oxidase

The magnesium ion in bovine heart cytochrome c oxidase can be depleted up to 75% by heat treatment of the enzyme at 43 degrees C followed by dialysis against EDTA buffer solution. The magnesium-depleted enzyme so obtained retains 40% of the activity of the native enzyme. This is the first attempt to deplete magnesium ion from bovine heart cytochrome c oxidase without denaturation of the protein. Magnesium depletion exposes at least one carboxyl group on subunit IV for labeling by N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide (NCD-4). The NCD-4 labeling of subunit IV of the magnesium-depleted enzyme is significantly enhanced relative to what is observed for the native and heat-treated oxidase, suggesting that the magnesium ion is located in subunit IV with at least one carboxyl ligand. By comparing the activity of the magnesium-depleted enzyme with that of a control sample of heat-treated oxidase, the influence of divalent magnesium on the activity of the enzyme is assessed.     

Extended metal environments of cytochrome c oxidase structures

The metals of the cytochrome c oxidase structures of the bovine heart mitochondrion (PDB code 1occ) and of the soil bacterium Paracoccus denitrificans (1arl) include a dicopper center (CuA), magnesium, two proximal hemes, a copper (CuB) atom, and a calcium. The mitochondrial structure also possesses a bound distant zinc ion. The extended environments of the metal sites are analyzed emphasizing residues of the second shell in terms of polarity, hydrophobicity, secondary structure, solvent accessibility, and H-bonding networks. A significant difference in the CuA metal environments concerns D-51 I in 1occ, absent from 1arl. The D-51 I appears to play an important role in the proton pumping pathway. Our analysis uncovers several statistically significant residue clusters, including a cysteine-histidine-tyrosine cluster overlapping the CuA-Mg complex; a histidine-acidic cluster enveloping the environment of Mg, the two hemes, and CuB; and on the protein surface a mixed charge cluster, which may help stabilize the quaternary structure and/or mediate docking to cytochrome c. These clusters may constitute possible pathways for electron transfer, for O2 diffusion, and for H2O movement. Many hydrogen bonding relations along the interface of subunits I and II demarcate this surface as a potential participant in proton pumping.     

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.     

Proton uptake controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the "peroxy" to "oxoferryl" transition, internal transfer of the fourth electron from CuA to heme a has the same rate as proton uptake from the bulk solution (8,000 s-1). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a CuA ligand, was replaced by leucine, which resulted in an increased redox potential of CuA. During reaction of the reduced mutant enzyme with O2, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s-1), whereas electron transfer from CuA to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from CuA to heme a were blocked, the results from this study show that the CuA --> heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a3-CuB before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.     

Involvement of cytochrome c oxidase subunit III in energy coupling

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.     

A microtiter plate assay for cytochrome c oxidase in permeabilized whole cells

A new mouse retinal degeneration that appears to be an excellent candidate for modeling human retinitis pigmentosa is reported. In this degeneration, called rd-3, differentiation proceeds postnatally through 2 weeks, and photoreceptor degeneration starts by 3 weeks. The rod photoreceptor loss is essentially complete by 5 weeks, whereas remnant cone cells are seen through 7 weeks. This is the only mouse homozygous retinal degeneration reported to date in which photoreceptors are initially normal. Crosses with known mouse retinal degenerations rd, Rds, nr, and pcd are negative for retinal degeneration in offspring, and linkage analysis places rd-3 on mouse chromosome 1 at 10 +/- 2.5 cM distal to Akp-1. Homology mapping suggests that the homologous human locus should be on chromosome 1q.     

Diazene--a not so innocent ligand for the binuclear center of cytochrome c oxidase

Diazene reacts rapidly with cytochrome c oxidase to reduce cytochrome a and CuA and to form a charge-transfer complex with ferric cytochrome a3; the diazene may serve to bridge the heme iron of this cytochrome and CuB. The complex is characterized by an intense, optically active absorbance located at 847 nm. A similar band had been observed previously upon reduction of cytochrome oxidase with hydrazine [Markossian, K. A., Paitian, N. A., and Nalbandyan, R. M. (1983) FEBS Lett. 156, 235-238], but it appears that this band is actually due to the diazene produced as a result of the oxidation of the hydrazine that occurs in this process. A similar diazene to iron charge-transfer band is found following the reaction of diazene with ferric horseradish peroxidase and with hemin chloride but not with met-myoglobin.     

Tissue-specific regulation of cytochrome c oxidase efficiency by nucleotides

Cytochrome c oxidase from bovine heart and liver was reconstituted in liposomes in the absence or presence of nucleotides. Intraliposomal ADP, and to a smaller extent intraliposomal ATP, increased the respiratory activity of the heart but not of the liver isozyme under uncoupled but not under coupled conditions, leading to increased respiratory control ratios. In a preceding publication [Anthony, G., Reimann, A., & Kadenbach, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 1652-1656], the stimulatory effect of intraliposomal ADP could be related to interaction with the matrix domain of subunit VIa-h (heart type). The data suggest a regulatory effect of matrix nucleotides in heart and skeletal muscle mitochondria on the efficiency of energy transduction in COX.     

Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen

The reduction of dioxygen to water by cytochrome c oxidase was monitored in the Soret region following photolysis of the fully reduced CO complex. Time-resolved optical absorption difference spectra collected between 373 and 521 nm were measured at delay times from 50 ns to 50 ms and analyzed using singular value decomposition and multiexponential fitting. Five processes were resolved with apparent lifetimes of 0.9 micros, 8 micros, 36 micros, 103 micros, and 1.2 ms. A mechanism is proposed and spectra of intermediates are extracted and compared to model spectra of the postulated intermediates. The model builds on an earlier mechanism that used data only from the visible region (Sucheta et al. (1997) Biochemistry 36, 554-565) and provides a more complete mechanism that fits results from both spectral regions. Intermediate 3, the ferrous-oxy complex (compound A) decays into a 607 nm species, generally referred to as P, which is converted to a 580 nm ferryl form (Fo) on a significantly faster time scale. The equilibrium constant between P and Fo is 1. We propose that the structure of P is a3(4+)=O CuB2+-OH- with an oxidizing equivalent residing on tyrosine 244, located close to the binuclear center. Upon conversion of P to Fo, cytochrome a donates an electron to the tyrosine radical, forming tyrosinate. Subsequently a proton is taken up by tyrosinate, forming F(I) [a3(4+)=O CuB2+-OH- a3+ CuA+]. This is followed by rapid electron transfer from CuA to cytochrome a to produce F(II) [a3(4+)=O CuB2+-OH- a2+ CuA2+].     

Essential role of mitochondrially encoded large rRNA for germ-line formation in Drosophila embryos

In Drosophila, pole cells, the progenitors of the germ line, are induced by the factors localized in the posterior pole region of oocytes and cleavage embryos, or germ plasm. Polar granules in germ plasm are electron-dense structures and have been proposed to contain factors essential for pole cell formation. Mitochondrially encoded large ribosomal RNA (mtlrRNA) has been identified as a component of polar granules. We previously have shown that mtlrRNA is able to rescue embryos that fail to form pole cells as a result of UV irradiation. However, there is a possibility that the function of mtlrRNA is limited to UV-irradiated embryos, and the question of whether mtlrRNA is required for the normal pathway leading to pole cell formation remains unanswered. In this study, we report that the reduction of mtlrRNA in germ plasm by injecting anti-mtlrRNA ribozymes into cleavage embryos leads to their inability to form pole cells. Other components of germ plasm, namely oskar mRNA, germ cell-less mRNA, and Vasa and Tudor proteins appear to be unaffected in these ribozyme-injected embryos. These results support an essential role for mtlrRNA in pole cell formation. We propose that mitochondrially encoded molecules participate in a key event in early cell-type specification.     

A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata

Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases. The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione. Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert. One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides. By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated. Cf21 proved to be a moderately efficient peroxidase with broad specificity. The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively. The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1. This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.