Bioenergetics at extreme temperature: Thermus thermophilus ba(3)- and caa(3)-type cytochrome c oxidases

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba(3)- and caa(3)-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa(3)-oxidase subunits having been initially 'fused'. Coupled with sequence analysis, the ba(3)-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate-enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included.     

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H⁺ and e^- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba₃-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O₂-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu_B atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe_a3 and Cu_B atoms that is best modeled as peroxide. The structure of ba₃-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba₃-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.     

Transferable Denitrification Capability of Thermus thermophilus

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N₂O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.     

The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba₃ (Tt ba₃) cytochrome oxidase revealed that O₂ binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010–21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the Cu_B⁺-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba₃ (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380–1390) affects O₂ and NO binding and the ability of Cu_B to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba₃ with O₂ were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O₂ and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba₃. The rate of O₂ and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived Cu_B⁺-CO complex did not prevent O-O bond splitting and the resulting P_M formation, which was significantly faster (5–10 times) than in the bovine heart enzyme. We propose that O₂ binding to heme a₃ in Tt ba₃ causes CO to dissociate from Cu_B⁺ in a concerted manner through steric and/or electronic effects, thus allowing Cu_B⁺ to act as an electron donor in the mixed-valence enzyme. The significantly faster O₂ binding and O-O bond cleavage in Tt ba₃ compared to analogous steps in the aa₃ oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.     

Anaerobic Growth,a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles

Despite the fact that the extreme thermophilic bacteria belonging to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that this nar operon can be transferred by conjugation to an aerobic Thermus strain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives of Escherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative plasmid integrated into the chromosome. First, the nar operon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of the nar cluster. Finally, the mating time required for the transfer of the nar operon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream of nar.

Most extreme thermophiles that live in geothermal environments are strict anaerobes (3, 11) as a consequence of the adaptation to the low solubility of oxygen at these temperatures. However, members of the genus Thermus constitute an exception to this general rule, being described taxonomically as strictly aerobic chemorganotrophs (2).However, we recently showed that one of the most thermophilic isolates of this genus, Thermus thermophilus HB8, was able to grow anaerobically when nitrate was present in the medium. Biochemical and genetic evidence demonstrated that this ability was related to the synthesis of a membrane-bound respiratory nitrate reductase complex whose protein components, the α (NarG; 136 kDa), β (NarH; 57 kDa), and γ (NarI; 28 kDa) subunits, were homologous (about 48 to 50% sequence identity) to those from mesophilic facultative anaerobes (e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced under low oxygen concentrations when nitrate was present (21). In contrast to those described for most nitrate reducers, the product of nitrate respiration was secreted to the growth medium through an unknown transporter.We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains HB8 and HB27 of T. thermophilus is the absence of plasmids from the latter, the possibility that the nar operon could be encoded by a transferable genetic element, such as a plasmid, was considered.In this article, we analyze this possibility and demonstrate that the ability to grow by nitrate respiration can be transferred to the aerobic strain T. thermophilus HB27 by conjugation. We also relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal genes in a time-dependent way.     

Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments

Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa₃- and cbb₃-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb₃-type oxidase is the predominant system for respiration of oxygen (O₂), especially when O₂ is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb₃-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa₃-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb₃- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa₃-type oxidase and switch of the regulatory system for respiration.     

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.     

Cytochromecaa 3 from the thermophilic bacteriumThermus thermophilus: A member of the heme-copper oxidase superfamily

The subject of this short review is the cytochromec oxidase (caa ₃) from the thermophilic bacteriumThermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing thatThermus cytochromecaa ₃ is abona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Spectroscopic and Kinetic Investigation of the Fully Reduced and Mixed Valence States of ba3-Cytochrome c Oxidase from Thermus thermophilus: A FOURIER TRANSFORM INFRARED (FTIR) AND TIME-RESOLVED STEP-SCAN FTIR STUDY*

The complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. Cytochrome c oxidase reduces O₂ to H₂O, a reaction coupled to proton translocation across the membrane. Therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers. In this study Fourier transform infrared (FTIR) and time-resolved step-scan FTIR spectroscopy have been applied to study the fully reduced and mixed valence states of cytochrome ba₃ from Thermus thermophilus. We used as probe carbon monoxide (CO) to characterize both thermodynamically and kinetically the cytochrome ba₃-CO complex in the 5.25–10.10 pH/pD range and to study the reverse intramolecular electron transfer initiated by the photolysis of CO in the two-electron reduced form. The time-resolved step-scan FTIR data revealed no pH/pD dependence in both the decay of the transient Cu_B¹⁺-CO complex and rebinding to heme a₃ rates, suggesting that no structural change takes place in the vicinity of the binuclear center. Surprisingly, photodissociation of CO from the mixed valence form of the enzyme does not lead to reverse electron transfer from the reduced heme a₃ to the oxidized low-spin heme b, as observed in all the other aa₃ and bo₃ oxidases previously examined. The heme b-heme a₃ electron transfer is guaranteed, and therefore, there is no need for structural rearrangements and complex synchronized cooperativities. Comparison among the available structures of ba₃- and aa₃-cytochrome c oxidases identifies possible active pathways involved in the electron transfer processes and key structural elements that contribute to the different behavior observed in cytochrome ba₃.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

The roles of Rhodobacter sphaeroides copper chaperones PCuAC and Sco (PrrC) in the assembly of the copper centers of the aa3-type and the cbb3-type cytochrome c oxidases

The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa₃-type CcO containing a di-copper Cu_A center and mono-copper Cu_B, plus a cbb₃-type CcO that contains Cu_B but lacks Cu_A. Three copper chaperones are located in the periplasm of R. sphaeroides, PCu_AC, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu_B of the aa₃-type but not the cbb₃-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu_A assembly in mitochondria and bacteria, and with Cu_B assembly of the cbb₃-type CcO. PCu_AC is present in many bacteria, but not mitochondria. PCu_AC of Thermus thermophilus metallates a Cu_A center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa₃- and cbb₃-type CcOs of R. sphaeroides has been examined in strains lacking PCu_AC, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu^2 +. The absence of PrrC has a greater effect than the absence of PCu_AC and PCu_AC appears to function upstream of PrrC. Analysis of purified aa₃-type CcO shows that PrrC has a greater effect on the assembly of its Cu_A than does PCu_AC, and both chaperones have a lesser but significant effect on the assembly of its Cu_B even though Cox11 is present. Scenarios for the cellular roles of PCu_AC and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu_A of the aa₃-type CcO and to Cu_B of the cbb₃-type CcO, while the predominant role of PCu_AC may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis

The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.     

Kinetic studies of the reactions of O2 and NO with reduced Thermus thermophilus ba3 and bovine aa3 using photolabile carriers

The reactions of molecular oxygen (O₂) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba₃ and bovine heart aa₃ were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O₂ and NO complexes. The binding of O₂/NO to reduced ba₃ in the absence of CO occurs with a second-order rate constant of 1 × 10⁹ M^? 1 s^? 1. This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O₂/NO binding in ba₃ is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O₂ and NO access to the active site in Tt ba₃, and that traditional CO flow-flash experiments do not accurately reflect the O₂ and NO binding kinetics in ba₃. We suggest that in ba₃ the binding of O₂ (NO) to heme a₃^2 + causes rapid dissociation of CO from Cu_B⁺ through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu_B⁺. These findings indicate that structural differences between Tt ba₃ and the bovine aa₃ enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ (ba₃) of Thermus thermophilus (T. thermophilus) is a member of the heme–copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a₃) and a copper (Cu_B). The heme–copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba₃ with O₂ using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~ 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s^? 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O₂ binds rapidly to heme a₃ in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O₂ binds transiently to a trap, likely Cu_B, prior to its migration to heme a₃ for the oxidative reaction, highlighting the critical role of Cu_B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.