Pulsed cytochrome c oxidase from the thermophilic bacterium PS3.

A caa3-type terminal cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 containing three subunits showed conversion from resting into pulsed form. Upon pulsing (reduction and re-oxidation), the cytochrome c oxidase activity increased over 10-fold. This enhanced activity of the pulsed enzyme gradually decayed. Addition of phospholipids, necessary for the enzyme activity, did not affect this decay process. Small changes in the absorption spectrum were observed for the resting-into-pulsed transition and for H2O2 ligation to the pulsed enzyme. The e.p.r. spectrum of the resting enzyme was very similar to that of mitochondrial enzyme, but the transient g = 5, 1.78 and 1.69 set of e.p.r. signals, associated with the pulsed bovine heart oxidase, were not observed in the case of pulsed bacterium-PS3 enzyme.     

Resonance Raman study of cytochrome aa3 from Sulfolobus acidocaldarius.

The single subunit terminal oxidase of Sulfolobus acidocaldarius, cytochrome aa3, was studied by resonance Raman spectroscopy. Results on the fully oxidized, the fully reduced, and the reduced carbon monoxide complex are reported and compared with those of eucaryotic cytochrome oxidase. It is shown that in both redox states the hemes a and a3 are in the six-coordinated low-spin and six-coordinated high-spin configuration, respectively. The resonance Raman spectra reveal far-reaching similarities of this archaebacterial with mammalian or plant enzymes except for the reduced form of heme a. The formyl substituent of this heme appears above 1640 cm-1, ruling out significant hydrogen bonding interactions which is in sharp contrast to beef heart cytochrome oxidase. In addition, frequency upshifts of the marker bands v4 and v2 are noted indicating differences in the electron density distribution within the molecular orbitals of the porphyrin.     

A cytochrome o-type oxidase of the thermophilic bacterium PS3 grown under air-limited conditions

A cytochrome o-type oxidase from the thermophilic bacterium PS3 grown under air-limited conditions was purified by ion-exchange chromatography in the presence of a non-ionic detergent. The enzyme was composed of three subunits (60, 30, and 16 kDa) and seemed to contain two molecules of heme b as prosthetic groups. It contained no detectable copper. The reduced enzyme showed absorption bands at 426 and 558.5 nm, and a characteristic spectral change upon binding CO. It oxidized several cytochromes c and artificial dyes such as N,N,N',N'-tetramethyl-p-phenylenediamine and phenazonium methosulfate at appreciable rates. Its Km for O2 was low (0.09 microM). It was capable of transmembrane electron transfer, because when reconstituted into liposomes, it generated a membrane potential upon oxidation without pumping protons.     

Reaction of caa3-type terminal cytochrome oxidase from the thermophilic bacterium PS3 with oxygen and carbon monoxide at low temperatures.

Reaction of O2 and CO with a caa3-type terminal cytochrome oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 grown with high aeration was studied at low temperatures. The CO recombination at the temperature range studied (-50 degrees C to -80 degrees C) followed first-order kinetics with an activation energy of 29.3 kJ/mol (7.0 kcal/mol). In the presence of O2 at -113 degrees C the photolysed reduced form binds O2 to form an 'oxy' intermediate similar to Compound A. At a higher temperature (-97 degrees C) another intermediate, similar to Compound B, is formed as a result of electron transfer from the enzyme to the liganded O2.     

Proton transport by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes

Cytochrome oxidase from the thermophilic bacterium PS3 which contains three types of polypeptide subunits are reconstituted into liposomes by a freeze-thaw technique. The reconstituted enzyme caused acidification of the medium during cytochrome c oxidation with a stoichiometry of up to 0.8 H+/e. Uptake of K+ ions in the presence of valinomycin occurred with a stoichiometry between 1.5 and 2 K+/e. Dicyclohexylcarbodiimide inhibited the acidification and decreased the stoichiometry of K+ ion uptake to about 1 K+/e. This bacterial oxidase thus appears to be a proton pump with properties similar to the mitochondrial enzyme.     

Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3

Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a3 centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

Nucleotide sequence of the gene coding for cytochrome oxidase subunit I from the thermophilic bacterium PS3

The gene coding for cytochrome oxidase subunit I (COI) was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminal of the COI protein was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequence of COI protein is composed of 536 amino acid residues and its molecular mass is 59,510. The protein is clearly homologous to the corresponding subunit in the mitochondrial cytochrome oxidase and similarly appears to have 12 trans-membrane segments. The proposed ligands to two hemes (cytochrome aa3) and a copper atom (CuB) in this protein (Holm et al. (1987) EMBO J. 6, 2819-2823) are conserved in the sequence.     

Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Cytochrome oxidase from thermophilic bacterium PS3 contains a fourth protein subunit

Monoclonal antibodies prepared against subunits II and IV of beef heart cytochrome oxidase were found to cross-react with thermophilic bacterial PS3 oxidase. Each individual antibody affects the enzymatic activity. "Western" blot analyses showed that subunit II antibodies of beef heart recognized subunit II of PS3 and subunit IV antibody likewise recognized a fourth protein subunit on slab gels. This fourth subunit previously thought to be a contaminant or a degradation product has a molecular weight of about 10,500 on SDS-gels, and appears to exist in stoichiometric amount. We have extracted this subunit from slab gels and compared its amino acid composition with that of subunit III.     

Identification and properties of a quinol oxidase super-complex composed of a bc1 complex and cytochrome oxidase in the thermophilic bacterium PS3

Evidence for the presence of a quinol oxidase super-complex composed of a cytochrome bc1 complex and cytochrome oxidase in the respiratory chain of a Gram-positive thermophilic bacterium PS3 is reported. On incubation with an octyl glucoside-solubilized fraction of the total membranes of PS3 anti-serum against PS3 cytochrome oxidase gave an immunoprecipitate that showed both quinol-cytochrome c reductase and cytochrome c oxidase activities. When the cholate-deoxycholate and LiCl-treated membranes of PS3 were solubilized and subjected to ion-exchange chromatography in the presence of octaethyleneglycol dodecyl ether, most of the A-, B-, and C-type cytochromes were copurified as a peak having both quinol-cytochrome c reductase and cytochrome oxidase activities. The immunoprecipitate and quinol oxidase preparation contained hemes a, b, and c in a ratio of about 2:2:3, indicating the presence of one-to-one complex of cytochrome oxidase containing 2 hemes a and one heme c, and a bc1 complex containing 2 hemes b and 2 hemes c. Gel electrophoresis in the presence of dodecyl sulfate showed that the immunoprecipitate and quinol oxidase preparation were composed of seven subunits; those of 51 (56-kDa), 38, and 22 kDa for cytochrome oxidase and those of 29, 23, 21, and 14 kDa for the bc1 complex. The 38-, 29-, and 21 kDa components possessed covalently bound heme c. The apparent molecular mass of the super complex was estimated to be as 380 kDa by gel filtration.     

Haem O2 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3.

Thermophilic bacterium PS3 cultured under slightly air-limited conditions showed a mitochondrion-like cytochrome pattern similar to that in vigorously aerated cells, but an o-type cytochrome replaced cytochrome a3 as the CO-binding centre. Cytochrome cao-type oxidase was purified from the cell membranes by almost the same procedure as used for cytochrome caa3. The turnover number of cytochrome cao was higher than that of cytochrome caa3, but the Km's of the two enzymes for cytochrome c and O2 were almost the same. Gel electrophoresis in the presence of sodium dodecyl sulfate gave bands of four subunits at the identical positions both for cytochrome cao and cytochrome caa3. Cytochrome cao contained a novel kind of haem in addition to haems C and A. This novel haem is likely to be haem O, very recently found as the chromophore of the cytochrome bo complex in Escherichia coli. These data suggest that cytochrome cao is an alternative form of cytochrome c oxidase (cytochrome caa3), in which the cytochrome a3 centre of the enzyme is replaced with cytochrome o.     

Sulfite oxidation catalyzed by aa(3)-type cytochrome c oxidase in Acidithiobacillus ferrooxidans

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa(3)-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the β-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa(3)-type cytochrome c oxidase. This is the first report to indicate that aa(3)-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.     

Some properties of Nitrosomonas europaea cytochrome c oxidase (aa3-type) which lacks CuA

From Nitrosomonas europaea which had been cultivated in a medium deficient in copper, cytochrome c oxidase (aa3-type) which did not have CuA was purified. The oxidase did not show the 830-nm peak and its ESR spectrum differed greatly from that of the normal enzyme, which has two copper atoms, CuA and CuB, per molecule. However, the oxidase which did not have CuA showed almost the same cytochrome c oxidizing activity as the normal oxidase.     

Resonance Raman studies of CuA-modified cytochrome c oxidase

Modification of the CuA site in mammalian cytochrome c oxidase has been used to elucidate the functional role of this center in the catalytic cycle of the enzyme. Both heat treatment in detergents and chemical modification by p-(hydroxymercuri)benzoate (pHMB) convert CuA to a lower potential type II center and effectively remove the site from the electron-transfer pathway during turnover. In this study, resonance Raman spectroscopy has been employed to investigate the effects of these CuA modifications on the heme active sites. The Raman data indicate some environmental perturbation of the heme a3 chromophore in the modified derivatives. Only pHMB modification and SB-12 heat treatment produced significant effects in the Raman spectra of the fully reduced enzyme. These perturbations are much less evident in the spectra obtained within 10 ns of CO photolysis from the fully reduced species of the modified enzymes. Transient Raman studies further indicate that the half-time for CO religation in the modified enzymes is quite similar to that of the native protein.     

Molecular and enzymatic properties of "cytochrome aa3"-type terminal oxidase derived from Nitrobacter agilis

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Nitrobacter agilis to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed absorption peaks at 422, 598, and 840 nm in the oxidized form, and at 442 and 606 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 436 and 604 nm, and the latter peak had a shoulder at 599 nm. The enzyme possessed 1 mol of heme a and 1.6 g-atom of copper per 41,000 g, and was composed of two kinds of subunits of 51,000 and 31,000 daltons. These results show that the structurally minimal unit of the enzyme molecule is composed of one molecule each of the two subunits and contains 2 molecules of heme a and 2-3 atoms of copper. the enzyme rapidly oxidized ferrocytochromes c of several eukaryotes as well as N. agilis ferrocytochrome c-552. The reactions catalyzed by the enzyme were strongly inhibited by KCN. The reduction product of oxygen catalyzed by the enzyme was concluded to be water on the basis of the ratio of ferrocytochrome c oxidized to molecular oxygen consumed.     

Resonance Raman applications in investigations of cytochrome c oxidase

Recent applications of resonance Raman (RR) spectroscopy in investigations of cytochrome c oxidase (CcO) are reviewed. Red-excited RR spectra for the fully oxidized "as-isolated" CcO tuned to the ligand-to-metal charge transfer absorption band at 655nm exhibit a Raman band at 755cm(-1) assignable to the ν(OO) stretching mode of a peroxide. Binding of CN(-) diminishes the RR band concomitant with the loss of the charge transfer absorption band. This suggests that a peroxide forms a bridge between heme a(3) and Cu(B). Time-resolved RR spectroscopy of whole mitochondria identified a band at 571cm(-1) arising from the oxygenated intermediate at Δt=0.4, 0.6 and 1.4ms. Bands at 804 and 780cm(-1) of the P and F intermediates were observed at Δt=0.6 and 1.4ms, respectively. The coordination geometries of the three intermediates are essentially the same as the respective species observed for solubilized CcO. However, the lifetime of the oxygenated intermediate in mitochondria was significantly longer than the lifetime of this intermediate determined for solubilized CcO. This phenomenon is due either to the pH effect of mitochondrial matrix, the effect of ΔpH and/or ΔΨ across the membrane, or the effect of interactions with other membrane components and/or phospholipids.     

Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.     

Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside

It is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl beta-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed "respiratory control"; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0 X F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.