Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus. How old is cytochrome oxidase?

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II. With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.     

The CO adduct of yeast cytochrome c oxidase. M?ssbauer and photolysis studies

M?ssbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The M?ssbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same M?ssbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO.     

M?ssbauer studies of cytochrome c' from Rhodospirillum rubrum.

Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with M?ssbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field M?ssbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.     

M?ssbauer study of CO dehydrogenase from Clostridium thermoaceticum

We have studied with M?ssbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials greater than -200 mV, all of the approximately 12 irons reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe4S4]1+ cluster. We have also observed the M?ssbauer spectrum (approximately 18% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65. Also present is a doublet (9% of Fe) with delta EQ = 2.90 mm/s and delta = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component seems to interact with a nickel site to form an EPR-silent complex with half-integral electronic spin. We have also characterized the iron environments of the S = 1/2 NiFeC complex. This complex contributes approximately 20% of the total M?ssbauer absorption when the EPR signal has approximately 0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the M?ssbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compositions in this enzyme premature.     

M?ssbauer study of cytochrome c2 from Rhodospirillum rubrum. Sign of the product gxgygz of some low spin ferric heme proteins.

We have studied cytochrome c2 from Rhodospirllum rubrum with M?ssbauer spectroscopy and electron paramagnetic resonance. The M?ssbauer data on the ferric protein, taken in external magnetic fields up to 50 kG, were analyzed within the framework of the ligand field model commonly used to evaluate low-spin ferric heme compounds. The data analysis shows that the determinant of the electronic g-tensor, i.e. the product gxgygz, is positive for cytochrome c2. We have reanalyzed published M?ssbauer data of some low-spin ferric heme proteins with respect to the sign of the g-tensor determinant. We find that gxgygz is also positive for the cytochromes c, bs, and P-450, and for chloroperoxidase.     

Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans?

We have used a gene replacement strategy to delete the previously isolated gene [(1987) EMBO J. 6, 2825-2833] for the cytochrome c oxidase subunit I from Paracoccus denitrificans. The resulting mutant was still able to synthesize active cytochrome c oxidase. This led us to look for another locus which could completely suppress the mutation. In this study we report the isolation of a second gene encoding subunit I. An open reading frame coding for cytochrome c 550 was found upstream from this gene. We suggest that there are isoenzymes of cytochrome c oxidase (cytochrome aa3) in this bacterium.     

Yeast cytochrome c oxidase: A model system to study mitochondrial forms of the haem–copper oxidase superfamily

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The purification and Mössbauer parameters of the haem undecapeptide of cytochrome c

A new method of preparing and purifying the haem undecapeptide of cytochrome c is reported. The Mössbauer spectra of solid samples, lyophilized at pH 7 from water, show mainly the presence of low-spin ferric iron, in contrast with earlier reports. No evidence of temperature dependent spin-spin equilibria was observed. A small proportion of the haem (～ 15%) inhabits an environment distinctly different from that of the majority. These observations are discussed.     

Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

M?ssbauer spectroscopy on nonequilibrium states of myoglobin: a study of r-t relaxation.

A frozen solution of 57Fe-enriched metmyoglobin was irradiated by x rays at 77 K. Mössbauer spectra showed a reduction of Fe(III) high spin by thermalized electrons and a production of a metastable Fe(II) low spin myoglobin complex with H2O at its sixth coordination site. The relaxation of the intermediate was investigated by Mössbauer spectroscopy as a function of temperature and time. The relaxation process starts above 140 K and is fully completed at approximately 200 K. At temperatures between 140 and 200 K, the relaxation lasts for hours and is nonexponential in time. Up to 180 K, the process can be described satisfactorily by a gamma distribution of activation enthalpies with an Arrhenius relation for the rate coefficient. The temperature and time dependence of the Mössbauer parameters indicates structural changes in the active center of the protein as early as 109 K that continue for several hours at higher temperatures. Above 180 K, structural rearrangements involving the whole protein molecule lead to a shift and narrowing of the barrier height distribution.     

A thermostable β-glucosidase isolated from a bacterial species of the genus Thermus

Summary An extremely thermophilic aerobic bacterium which produced -glucosidase was isolated from soil collected at the Yudanaka hot spring in Japan. It was identified as belonging to the genus Thermus. Production of -glucosidase by this bacterium was stimulated by the addition of cellobiose or laminaribiose to the medium. The optimum pH and temperature of the enzyme were 4.5–6.5 and 85° C respectively. The enzyme was stable in the pH range of 4.5–7.0 at 70° C for 2 h and the half-life at 75° C was 5 days. The K _m value of the enzyme for p-nitrophenyl--d-glucopyranoside, determined at 70° C in 0.1 M sodium phosphate buffer (pH 6.5), was 0.28 mM while the K _m was 2.0 mM for cellobiose. The enzyme effectively hydrolysed cellobiose at 70° C and the conversion yields of cellobiose to glucose were 95%, 93% and 90% at substrate concentrations of 5%, 10% and 15%, respectively.     

The complex of cytochrome c and cytochrome c peroxidase: the end of the road?

Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form a physiological complex in the inter-membrane space of yeast mitochondria, where CcP reduces hydrogen peroxide to water using the electrons provided by ferrous Cc. The Cc-CcP system has been a popular choice of study of interprotein biological electron transfer (ET) and in understanding dynamics within a protein-protein complex. In this review we have charted seven decades of research beginning with the discovery of CcP and leading to the latest functional and structural work, which has clarified the mechanism of the intermolecular ET, addressed the putative functional role of a low-affinity binding site, and identified lowly-populated intermediates on the energy landscape of complex formation. Despite the remarkable attention bestowed on this complex, a number of outstanding issues remain to be settled on the way to a complete understanding of Cc-CcP interaction.     

Does the peroxide compound of cytochrome oxidase contain a ferryl iron?

The reaction of peroxide with cytochrome oxidase generates a peroxide compound having a Soret maximum at 428 nm. X-ray absorption spectroscopy analysis of the local structure of the active site iron shows marked similarity to that of the cytochrome c peroxidase intermediate Compound ES, which contains a short iron to proximal nitrogen distance compared to globins. Reductive titration of the 580 nm band of this compound indicates that the iron is one oxidizing equivalent above the resting oxidized form. These results support the presence of a ferryl iron (Fe(IV) = O) in the peroxide compound similar to that found for the peroxidases.     

The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage?

Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.     

Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe M?ssbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.     

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.