Cytochrome Ba3 Research Articles

Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.

Mutations of the Weak Axial Ligand in the Thermus CuA Center Modulates Its Electronic Structure

Here we report the first stable, axial ligand mutations, Met160Gln and Met160Glu, of a Cu_A center in the Thermus thermophilus (TtIICu_A) fragment of cytochrome ba3.2a The binuclear, delocalized (S = 1/2) Cu_A center serves as the initial electron acceptor in cytochrome c oxidases (COX) and nitrous oxide reductase (N_2OR). The Cu_A domains and the type 1 (T1) blue copper centers are members of the cupredoxin superfamily of electron transfer (ET) proteins. The Cu_A centers have two bridging Cys thiolates, two terminal His imidazole ligands, and one weak axial ligand to each copper, a Met thioether or the peptide carbonyl of Glu (Figure 1). The weak axial ligands give each Cu a distorted tetrahedral geometry, similar to the T1 centers.

The active site structure of ba3 oxidase from Thermus thermophilus studied by resonance raman spectroscopy.

The ba3 cytochrome oxidase from Thermus thermophilus was studied by resonance Raman spectroscopy. The component spectra of both heme groups were determined by using different excitation wavelengths. In the ferric state the heme a3 group reveals resonance Raman marker bands characteristic for two high spin species with the heme iron in an in-plane and an out-of-plane configuration that reflects a coordination equilibrium. This equilibrium obviously results from protonation of one of the axial ligands that is ascribed to a hydroxide. Coordination by its protonated form, a water molecule, may be too weak to keep the heme iron in the porphyrin plane. The corresponding Fe-OH2 stretching mode was attributed to a weak H/D-sensitive band at 464 cm(-1). The coordination equilibrium not only depends on the pH but is also affected by the buffer, the salt concentration, and the binding of the natural redox partner cytochrome c552. These changes of the coordination equilibrium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a relay of hydrogen bonds. Environmental changes at the catalytic site are sensitively reflected by the formyl stretching of heme a3. The unique structural properties of the ba3 oxidase may be related to the unusual proton pump efficiency and heme a3 redox potential.

Cloning and Expression in Escherichia coli of the Cytochrome c552 Gene from Thermus thermophilus HB8: EVIDENCE FOR GENETIC LINKAGE TO AN ATP-BINDING CASSETTE PROTEIN AND INITIAL CHARACTERIZATION OF THE cycA GENE PRODUCTS

We report sequence of Thermus thermophilus HB8 DNA containing the gene (cycA) for cytochrome c552 and a gene (cycB) encoding a protein homologous with one subunit of an ATP-binding cassette transporter. The cycA gene encodes a 17-residue N-terminal signal peptide with following amino acid sequence identical to that reported by (Titani, K., Ericsson, L. H., Hon-nami, K., and Miyazawa, T. (1985) Biochem. Biophys. Res. Commun. 128, 781-787). A modified cycA was placed under control of the T7 promoter and expressed in Escherichia coli. Protein identical to that predicted from the gene sequence was found in two heme C-containing fractions. Fraction rC552, characterized by an alpha-band at 552 nm, contains approximately 60-70% of a protein highly similar to native cytochrome c552 and approximately 30-40% of a protein that contains a modified heme. Cytochrome rC552 is monomeric and is an excellent substrate for cytochrome ba3. Cytochrome rC557 is characterized by an alpha-band at 557 nm, contains approximately 90% heme C and approximately 10% of non-C heme, exists primarily as a homodimer, and is essentially inactive as a substrate for cytochrome ba3. We suggest that rC557 is a "conformational isomer" of rC552 having non-native, axial ligands to the heme iron and an "incorrect" protein fold that is stabilized by homodimer formation.

Cyanide binding and active site structure in heme-copper oxidases: normal coordinate analysis of iron-cyanide vibrations of a3(2+)CN- complexes of cytochromes ba3 and aa3.

The cyanide isotope-sensitive low-frequency vibrations of ferrous cyano complexes of cytochrome a3 are studied for cytochrome ba3 from Thermus thermophilus and cytochrome aa3 from bovine heart. Cyanide complexes of ba3 display three isotope sensitive frequencies at 512, 485, and 473 cm-1. The first is primarily an Fe-C stretching motion, whereas the lower wavenumber modes are bending motions. These iron-cyanide vibrations are independent of the redox levels of the other metal centers in the protein. On the other hand, the fully reduced bovine derivative complexed with cyanide gives rise to a bending vibration at 503 cm-1 and a stretching vibration at 469 cm-1. That is, the ordering of the stretching and bending frequencies is reversed from that of the bacterial protein. These results are analyzed by normal coordinate calculations to obtain comparative models for the binuclear O2 reducing site of the two proteins. We find that the observed frequencies are consistent with a linear Fe-C-N group and larger Fe-C stretching force constant (2.558 mdyn/A) for ba3 and a slightly bent Fe-C-N group (angle approximately 170 degrees) and a smaller Fe-C stretching force constant (2.335 mdyn/A) for aa3. Thus, there are significant differences in the interaction of cyanide with ferrous a3 in the two proteins that are most likely caused by a weaker proximal histidine interaction and stronger peripheral heme electron withdrawing effects in ba3. Possible sources of these protein-induced effects are discussed. Using the analysis developed here, comparison of the FeCN stretching and bending frequencies of the ferrous bovine a3-CN complex to those obtained from the ferric a3-CN complex suggests that upon conversion of the resting to the fully reduced protein, a conformational change occurs that constrains the ligand binding site.

Cytochrome ba3 from Natronobacterium pharaonis--an archaeal four-subunit cytochrome-c-type oxidase.

Cytochrome ba3, a terminal oxidase was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis. NH2-terminal sequence information of two subunits with apparent molecular masses of 40 and 36 kDa was used to generate a DNA probe by polymerase chain reaction. Cloning and sequencing of two overlapping genomic fragments revealed four genes forming a transcriptional unit. The policystronic messenger RNA of this cbaDBAC gene locus was identified by RNA analysis. The genes cbaC and cbaD code for small hydrophobic peptides with 81 and 54 amino acids. The genes cbaB and cbaA code for cytochrome oxidase subunit II (calculated molecular mass = 18.6 kDa) and I (calculated molecular mass = 63.8 kDa) respectively. Five potential CuA ligands for subunit II and six His residues for subunit I located in conserved positions indicate cytochrome ba3 to be a c-type oxidase. Sequence comparison and phylogenetic analysis place the natronobacterial enzyme together with the archaeal quinol oxidase SoxABCD from Sulfolobus acidocaldarius and the eubacterial ba3-type oxidase from Thermus thermophilus into a distinct evolutionary group. All three members are missing residues which are functionally important for vectorial proton translocation. The four-subunit enzyme complex was also identified on the protein level using chromatographic buffers containing ethylene glycol for purification.

Electron transfer proteins from the haloalkaliphilic archaeon Natronobacterium pharaonis: possible components of the respiratory chain include cytochrome bc and a terminal oxidase cytochrome ba3.

Natronobacterium pharaonis, an aerobic haloalkaliphilic archaebacterium, expresses high concentrations of redox proteins as do alkaliphilic eubacteria. The first redox protein characterized from N. pharaonis was halocyanin [Scharf, B., & Engelhard, M. (1993) Biochemistry 32, 12894-12900], a small blue copper protein. It is a peripheral membrane protein and is conjectured to function in a manner similar to plastocyanin. In the present work, the respiratory chain is further elucidated and the purification and characterization of the most abundant components cytochrome bc and cytochrome ba3 from the membrane fraction are described. The cytochrome bc complex consists of a 14 and an 18 kDa subunit in a 1:1 ratio, with heme c bound to the larger polypeptide. An Fe-S subunit similar to that found in eukaryotic bc complexes has not yet been identified. The second membrane complex carries two different heme groups of the ba3-type as well as copper. It contains two subunits of 36 and 40 kDa. This cytochrome ba3 binds carbon monoxide, a feature common to terminal oxidases. There is no spectroscopic evidence for a second terminal oxidase; hence, under the growth conditions chosen the respiratory chain of N. pharaonis appears to be unbranched. In addition to these cytochromes, a succinate dehydrogenase which is solubilized from the membrane by detergents was isolated. A cytochrome c which was isolated from the cytosol has an unusually high molecular weight and a redox potential of -142 mV. A second cytosolic protein, ferredoxin, was purified to homogeneity. A comparison of the redox potentials of the isolated proteins with those obtained from the native membrane allows the construction of a possible electron transfer chain.

Reaction of mercurials with the CuA center in the soluble fragment of cytochrome ba 3 subunit II from Thermus thermophilus

Optical absorption, EPR and electrospray ionization mass spectrometries were used to characterize a stoichiometric reaction between mercurials and the soluble ba 3–CuA protein from Thermus thermophilus. Either one Hg(II) or two RHg(II)ions react(s) to destroy the unique spectral properties of the CuA center. EPR spectra of the resulting product indicate that one Cu from the binuclear CuA center is released into the medium as a Type 2 Cu(II) while the other remains EPR silent. Mass spectra indicate that either one Hg(II) or two RHg(II) ions remain(s) bound to the protein along with one Cu, which is assumed to be a Cu(I) ion. The latter is slowly released from the protein under aerobic conditions as additional Type 2 Cu(II), and this process is catalyzed by fungal laccase, which serves as a strong one-electron oxidant.

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

The electrochemistry of a water-soluble fragment from the CuA domain of Thermus thermophilus cytochrome ba3 has been investigated. At 25 °C, CuA exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)2Cu]+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)2Cu]+/0 electron transfer. An irreversible oxidation of [Cu(Cys)2Cu]+ at 1 V vs NHE confirms that the CuII:CuII state of CuA is significantly destabilized relative to the CuII state of analogous blue-copper proteins.

Biochemical and spectroscopic properties of the four-subunit quinol oxidase (cytochrome ba 3) from Paracoccus denitrificans

The ba 3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079–23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

Electron paramagnetic resonance studies of the soluble CuA protein from the cytochrome ba3 of Thermus thermophilus

The electron paramagnetic resonance (EPR) spectrum of the binuclear CuA center in the water-soluble subunit II fragment from cytochrome ba3 of Thermus thermophilus was recorded at 3.93, 9.45, and 34.03 GHz, and the EPR parameters were determined by computer simulations. The frequency and M1 dependence of the linewidth was discussed in terms of g strain superimposed on a correlation between the A and g values. The g values were found to be gx = 1.996, gy = 2.011, gz = 2.187, and the two Cu ions contribute nearly equally to the hyperfine structure, with magnitude of Ax magnitude of approximately 15 G, magnitude of Ay magnitude = 29 G, and magnitude of Az magnitude of = 28.5 G (65Cu). Theoretical CNDO/S calculations, based on the x-ray structure of the Paracoccus denitrificans enzyme, yield a singly occupied antibonding orbital in which each Cu is pi*-bonded to one S and sigma*-bonded to the other. In contrast to the equal spin distribution suggested by the EPR simulations, the calculated contributions from the Cu ions differ by a factor of 2. However, only small changes in the ligand geometry are needed to reproduce the experimental results.

Electron-transfer studies with the Cu A domain of Thermus thermophilus cytochrome ba3

Flash photolysis has been used to initiate electron transfer from excited tris(2,2′-bipyridyl)ruthenium(II) to the Cu A site of a soluble domain from subunit II of Thermus thermophilus cytochrome ba 3. Luminescence quenching of the excited state of the ruthenium(II) complex was observed at low protein concentrations (20–200 μM Cu A domain), with second-order rate constants of 2.9 × 10 9 and 1.3 × 10 9 M −1 s −1 at low and high ionic strength, respectively. Transient absorption measurements demonstrate that 10–20% of the quenching arises from electron transfer (ET). At high protein concentrations (>250 μM Cu A) and low ionic strength (5 mM Tris, pH 8.1), the quenching rate saturates due to ground-state complex formation; a first-order rate constant of 1.5 × 10 5 s −1 was estimated for ET in the complex. Given the high driving forces involved ( ΔG° = 1.1 eV), it is possible that these ET reactions occur in the inverted driving-force regime. Spectroscopic measurements indicate that the T. thermophilus Cu A domain and horse heart cytochrome c form a complex at low ionic strength, with an apparent dissociation constant K d ∼ 5 μM.

Water-soluble, recombinant CuA-domain of the cytochrome ba3 subunit II from Thermus thermophilus.

Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

P-E3-19 Structure and reactions of the Cu A center of subunit II from cytochrome ba3 of Thermus thermophilus: Vänngård T, 1 Aasa R, 1 Karpefors M, 1 Larsson S, 2 Slutter CE, 3 Fee JA. 4 1 Dept. of Biochemistry & Biophysics, Göteborg Univ. (S), 2 Dept. of Physical Chemistry, Chalmers Univ. of Technology, Göteborg, (S), 3 Div. of Chemistry

P-E3-19 Structure and reactions of the Cu A center of subunit II from cytochrome ba3 of Thermus thermophilus: Vänngård T, 1 Aasa R, 1 Karpefors M, 1 Larsson S, 2 Slutter CE, 3 Fee JA. 4 1 Dept. of Biochemistry & Biophysics, Göteborg Univ. (S), 2 Dept. of Physical Chemistry, Chalmers Univ. of Technology, Göteborg, (S), 3 Div. of Chemistry

The CuA Center of a Soluble Domain from Thermus Cytochrome ba3. An NMR Investigation of the Paramagnetic Protein

The Cu_A center in subunit II of cytochrome c oxidase, the terminal enzyme of aerobic respiration, transfers electrons from cytochrome c to the proton-pumping machinery in subunit I. The unique electronic absorption and EPR spectra of Cu_A exclude it from classification with the well-studied biological copper centers. High-resolution X-ray structures of Cu_A-containing proteins reveal two copper atoms approximately 2.5 A apart, bridged by two cysteine sulfurs. Each Cu has a terminal histidine ligand and a weak ligand, methionine for one and a main chain carbonyl for the other. These structures are consistent with earlier EPR measurements and theoretical calculations, which predicted a highly delocalized mixed-valence [Cu(II),Cu(I)] Cu_A site. Here we report ^1H NMR measurements at 600 MHz on a soluble Cu_A domain from Thermus thermophilus cytochrome ba_3.

Molecular Genetic and Protein Chemical Characterization of the Cytochrome ba3 from Thermus thermophilus HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.

Multi-frequency EPR Evidence for a Binuclear Cu A Center in Cytochrome c Oxidase: Studies with a 63Cu- and 65Cu-Enriched, Soluble Domain of the Cytochrome ba3 Subunit II from Thermus thermophilus

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble Cu A-protein from the cytochrome ba 3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent g z ∼2.18 and g xy ∼2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that Cu A is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

A cytochrome ba3 functions as a quinol oxidase in Paracoccus denitrificans. Purification, cloning, and sequence comparison.

A quinol oxidase has been purified from the cytoplasmic membrane of Paracoccus denitrificans; its heme composition and CO binding properties identify it as a cytochrome ba3. On SDS gels, the purified enzyme complex is separated into five polypeptides. Using partial peptide sequence information for subunit II, the gene locus has been cloned and sequenced. In a typical operon pattern, four genes were identified: qoxA, -B, -C, and -D, coding for subunits II, I, III, and IV. DNA-derived amino acid sequence comparisons reveal extensive similarities to other members of the terminal oxidase superfamily.

Spectroscopic characterization of cytochrome ba3, a terminal oxidase from Thermus thermophilus: Comparison of the a3/CuB site to that of bovine cytochrome aa3

Unliganded and cyano derivatives of cytochrome ba3 from Thermus thermophilus have been examined by UV-vis, EPR, and resonance Raman spectroscopies. Species of cytochrome ba3 investigated include its resting, as-isolated, fully oxidized state, the fully reduced, unliganded enzyme, the one-electron-reduced cyano complex, the three-electron-reduced cyano complex, and the fully reduced cyano complex. Results are compared to those obtained from similar adducts of bovine cytochrome aa3, in particular, the fully reduced cyano complex. Our objective was to identify structural similarities and differences at the ligand-binding binuclear site of the two enzymes. We observed that the inner core skeletal vibrations of cytochrome a3 are the same for similar adducts of the bacterial ba3 and mammalian aa3, indicating similar spin and iron-porphyrin coordination properties resulting in comparable porphyrin core geometries. On the other hand, many of the vibrational frequencies associated with the formyl and vinyl peripheral substituents, and the outer pyrrole carbon atoms differ between the bovine and bacterial enzymes. Use of 57Fe labeled ba3 allows identification of two separate vFe-N(His) frequencies displayed by the fully reduced, unliganded cytochrome. These frequencies, occurring at 193 and 209 cm-1, are ascribed to distinct protein conformers, which are best evidenced by the Fe-N(His) vibrations. This result is again in contrast to the bovine enzyme which has been shown by others to display a single Fe-N(His) stretching frequency at 214 cm-1. The low-frequency Fea3(2+)-CN- vibrations of the three-electron and fully reduced cyano complexes of cytochrome ba3 are identified by using 15N and 13C isotopomers of CN-. These spectral signatures are identical to those reported earlier for the one-electron-reduced cyanide adduct (cytochrome a3 reduced), showing that the Fea3(2+)-CN- vibrational frequencies are independent of the redox states of the other three metal centers. Similarly, the CuB2+ EPR signatures appear similar in both the one-electron- and three-electron-reduced cyanide adducts. On the other hand, the electronic absorption spectra of ferrous alpha 3-CN- show systematic red-shifts of the alpha band as each of the other metal centers is reduced, and other, more subtle, differences in the electronic absorptions of the three-electron-reduced and four-electron-reduced cyanide adducts are revealed in the difference spectra. The relevance of these findings toward explaining the different cyanide binding and redox chemistry described herein and toward establishing the extent of structural analogy between the oxygen binding sites of the two proteins is discussed.

Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti.

Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o.