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Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O–O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O–O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion o...

After a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome c oxidase, we present the results of our earlier and recent density functional theory calculations for the dinuclear Fe-a3–CuB reaction center in this enzyme. A specific catalytic reaction wheel diagram is constructed from the calculations, based on the structures and relative energies of the intermediate states of the reaction cycle. A larger family of tautomers/protonation states is generated compared to our earlier work, and a new lowest-energy pathway is proposed. The entire reaction cycle is calculated for the new smaller model (about 185–190 atoms), and two selected arcs of the wheel are chosen for calculations using a larger model (about 205 atoms). We compare the structural and redox energetics and protonation calculations with available experimental data. The reaction cycle map that we have built is positioned for further improvement and testing against experiment., From density functional theory calculations on the dinuclear Fe-a3−CuB complex (DNC) in a type B bacterial cytochrome c oxidase, we construct a catalytic reaction wheel diagram for the full redox, O2 binding, and proton-transfer cycle. A new low-energy pathway is found. Strong shifts in the dipole moment direction/magnitude over the cycle and associated energy differences indicate that the DNC plays a major role not only in the chemical reaction cycle but also in proton pumping.

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

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PMID: 22402225; The hydrophobically guided complex formation between the Cu(A) fragment from Thermus thermophilus ba(3) terminal oxidase and its electron transfer substrate, cytochrome c(552), was investigated electrochemically. In the presence of the purified Cu(A) fragment, a clear downshift of the c(552) redox potential from 171 to 111mV±10mV vs SHE' was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

We use a form of "freeze-trap, kinetic crystallography" to explore the migration of Xe atoms away from the dinuclear heme a(3)/Cu(B) center in Thermus thermophilus cytochrome ba(3) oxidase. This enzyme is a member of the heme-copper oxidase superfamily and is thus crucial for dioxygen-dependent life. The mechanisms involved in the migration of oxygen, water, electrons, and protons into and/or out of the specialized channels of the heme-copper oxidases are generally not well understood. Pressurization of crystals with Xe gas previously revealed a O(2) diffusion channel in cytochrome ba(3) oxidase that is continuous, Y-shaped, 18-20 Å in length and comprised of hydrophobic residues, connecting the protein surface within the bilayer to the a(3)-Cu(B) center in the active site. To understand movement of gas molecules within the O(2) channel, we performed crystallographic analysis of 19 Xe laden crystals freeze-trapped in liquid nitrogen at selected times between 0 and 480 s while undergoing outgassing at room temperature. Variation in Xe crystallographic occupancy at five discrete sites as a function of time leads to a kinetic model revealing relative degrees of mobility of Xe atoms within the channel. Xe egress occurs primarily through the channel formed by the Xe1 → Xe5 → Xe3 → Xe4 sites, suggesting that ingress of O(2) is likely to occur by the reverse of this process. The channel itself appears not to undergo significant structural changes during Xe migration, thereby indicating a passive role in this important physiological function.

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 (CuB). 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 CuB, prior to its migration to heme a3 for the oxidative reaction, highlighting the critical role of CuB in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba 3 -type oxygen reductase from Thermus thermophilus , leading from the propionates of heme a 3 to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a 3 , and for Glu126 II (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126 II , and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a 3 , was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a 3 or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a 3 is a good candidate to be the proton loading site.

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba 3 cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O 2 -carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O 2 -carrier with a 355 nm laser pulse and the fully reduced CO-bound ba 3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O 2 binding to heme a 3 at 90 μM O 2 occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b . The second-order rate constant of 1 × 10 9 M -1 s -1 for O 2 binding to ba 3 in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O 2 bond cleavage in ba 3 of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O 2 to the active site in ba 3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a 3 site in ba 3 , making the CO flow-flash method inapplicable.

The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B- and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D- and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba 3 oxygen reductase from Thermus thermophilus , a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O 2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the Cu B and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.

Bacterial heme−copper terminal oxidases react quickly with NO to form a heme−nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N2O. Previously, we characterized the heme a3−NO complex formed in cytochrome ba3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to CuB(I) to form an end-on NO−CuB or a side-on copper−nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme−copper active site. Here we present a comparative study with cytochrome bo3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N2O. The EPR and resonance Raman signatures of the heme o3−NO complex are comparable to those of the a3−NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o3−NO complex does not produce a CuB−nitrosyl complex, but that instead, the NO remai...

In respiring organisms cytochrome c oxidase catalyzes proton translocation coupled to the reduction of O2 to water (1, 2). Cytochrome ba3 oxidase is one of two heme-copper oxidases isolated from T. thermophilus (3). It contains the dinuclear CuA center; a six-coordinated, low-spin, (6cLS) heme-B; and a (usually) five-coordinated, high-spin (5cHS) heme-As in close proximity to CuB ((3, 4) and references therein). The latter (heme-As and CuB) constitute the bimetallic site, and it is widely agreed that dioxygen binds to this site as the first step in O2-reduction and proton pumping. Two crystal structures of native and recombinant, oxidized ba3-type cytochrome c oxidase have been solved at

A mechanism for proton pumping by the B-type cytochrome c oxidases is presented in which one proton is pumped in conjunction with the weakly exergonic, two-electron reduction of Fe-bound O 2 to the Fe-Cu bridging peroxodianion and three protons are pumped in conjunction with the highly exergonic, two-electron reduction of Fe(III)- (-)O-O (-)-Cu(II) to form water and the active oxidized enzyme, Fe(III)- (-)OH,Cu(II). The scheme is based on the active-site structure of cytochrome ba 3 from Thermus thermophilus, which is considered to be both necessary and sufficient for coupled O 2 reduction and proton pumping when appropriate gates are in place (not included in the model). Fourteen detailed structures obtained from density functional theory (DFT) geometry optimization are presented that are reasonably thought to occur during the four-electron reduction of O 2. Each proton-pumping step takes place when a proton resides on the imidazole ring of I-His376 and the large active-site cluster has a net charge of +1 due to an uncompensated, positive charge formally associated with Cu B. Four types of DFT were applied to determine the energy of each intermediate, and standard thermochemical approaches were used to obtain the reaction free energies for each step in the catalytic cycle. This application of DFT generally conforms with previously suggested criteria for a valid model (Siegbahn, P. E. M.; Blomberg, M. A. R. Chem. Rev. 2000, 100, 421-437) and shows how the chemistry of O 2 reduction in the heme a 3 -Cu B dinuclear center can be harnessed to generate an electrochemical proton gradient across the lipid bilayer.

In heme-copper oxidases, the correlation curve between the iron-CO and C-O stretching vibrational modes (ν(Fe-CO) and ν(C-O), respectively) is anomalous as compared to the correlation in other heme proteins. To extend the correlation curve, the resonance Raman (RR) and infrared (IR) spectra of the CO adducts of cytochrome ba3 (ba3) from Thermus thermophilus were measured. The RR spectrum has two strong ν(Fe-CO) lines (508 and 515 cm(-1)) and a very weak line at 526 cm(-1), and the IR spectrum has three ν(C-O) lines (1966, 1973, and 1981 cm(-1)), indicating the presence of multiple conformers. Employing photodissociation methods, the ν(Fe-CO) RR and ν(C-O) IR lines were assigned to each conformer, enabling the establishment of a reliable inverse correlation curve for the ν(Fe-CO) versus the ν(C-O) stretching frequencies. To determine the molecular basis of the correlation, a series of DFT calculations on 6-coordinate porphyrin-CO compounds and a model of the binuclear center of the heme-copper oxidases were carried out. The calculations demonstrated that the copper unit model caused significant mixing among porphyrin-CO molecular orbitals (MOs) that contribute to the Fe-C and C-O bonding interactions, and also indicated the presence of mixing between the d(z)(2) orbital of the copper and MOs that are responsible for the ν(Fe-CO) vs ν(C-O) inverse correlation. Together, the spectroscopic and DFT results clarify the origin of the anomaly of ν(Fe-CO) and ν(C-O) frequencies in the heme-copper oxidases, a long-standing issue.

The structure of the soluble Rieske protein from Thermus thermophilus has been determined at a resolution of 1.3 A at pH 8.5 using multiwavelength anomalous dispersion (MAD) techniques. This is the first report of a Rieske protein from a menaquinone-utilizing organism. The structure shows an overall fold similar to previously reported Rieske proteins. A novel feature of this crystal form appears to be a shared hydrogen between the His-134 imidazole ring ligated to Fe2 of the [2Fe-2S] cluster and its symmetry partner, His-134', one being formally an imidazolate anion, Fe2-(His-134)N(epsilon)(-)...H-N(epsilon')(His-134')-Fe2', in which crystallographic C(2) axes pass equidistant between N(epsilon)...N(epsilon') and normal to the line defined by N(epsilon)...N(epsilon'). This provides evidence for a stable, oxidized cluster with a His(-) ligand and lends support to a previously proposed mechanism of coupled proton and electron transfer. A detailed comparison of the Thermus Rieske protein with six other Rieske and Rieske-type proteins indicates: (a) The cluster binding domain is tightly conserved. (b) The 3-D structure of the 10 beta-strand fold is conserved, even among the most divergent proteins. (c) There is an approximately linear relation between acid-pH redox potential and number of H-bonds to the cluster. (d) These proteins have two faces, one points into the larger complex (bc(1), b(6)f, or other), is involved in the proton coupled electron transfer function, and is highly conserved. The second is oriented toward the solvent and shows wide variation in charge, sequence, length, hydrophobicity, and secondary elements in the loops that connect the beta-sheets.

Cytochrome rC(557) is an improperly matured, dimeric cytochrome c obtained from expression of the "signal peptide-lacking" Thermus thermophilus cycA gene in the cytoplasm of Escherichia coli. It is characterized by its Q(00) (or alpha-) optical absorption band at 557 nm in the reduced form (Keightley, J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., and Fee, J. A. (1998) J. Biol. Chem. 273, 12006-12016). We report results of a broad ranging, biochemical and spectral characterization of this protein that reveals the presence of a free vinyl group on the porphyrin and a disulfide bond between the protomers and supports His-Met ligation in both valence states of the iron. A 3-A resolution x-ray structure shows that, in comparison with the native protein, the heme moiety is rotated 180 degrees about its alpha,gamma-axis; cysteine 14 has formed a thioether bond with the 2-vinyl of pyrrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs in the native protein; and a cysteine 11 from each protomer has formed an intermolecular disulfide bond. Numerous, minor perturbations exist within the structure of rC(557) in comparison with that of native protein, which result from heme inversion and protein-protein interactions across the dimer interface. The unusual spectral properties of rC(557) are rationalized in terms of this structure.

We present in this work low-temperature visible absorption spectra for recombinant Thermus thermophilus cytochrome c552. The Q-band presents a remarkable splitting at low temperature. We performed quantum chemical calculations to evaluate quantitatively the effect of heme conformation, axial ligand, peripheral substituents and local electric fields on the electronic spectra. In an attempt to find correlation between protein structure and spectral splitting, we carried out the same calculations on three other cytochrome c's: horse heart, tuna heart, and yeast. The quantum chemical calculations were performed at the INDO level with extensive configuration interaction. The electric field at the heme pocket was included in the calculations through a set of point charges fitting the actual electric field. The results obtained show clearly that all mentioned effects contribute to the observed spectral splitting in a complex nonadditive way.

The ability of myoglobin (Mb) to reversibly bind O2 and other ligands has been well characterized. Mb also participates with a variety of redox metals to form metmyoglobin (metMb). By using an anaerobic stopped-flow device we have measured outer-sphere oxidation by [Fe(CN)6]3− of native sperm whale myoglobin, recombinant wild-type Mb, and a series of mutant Mb proteins in which the distal His-64 was changed to Gly, Phe, Leu or Val. Second-order rate constants for oxidation of mutant proteins are 10–15 times greater than for recombinant or native (kox ~ 106 M− s−). We attribute the reduced rate of oxidation of wild-type protein to a higher reorganization energy imposed by the presence of the unique water/His-64/heme interaction, which is absent in the mutant proteins.

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.

Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.

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.

Received September 8, 1997; accepted January 5, 1998 The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc\ or b6f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus. Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al., 1996 Structure 4, 567-579).

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 oxidase is the terminal oxidase in both prokaryotic and eukaryotic cells and is responsible for the generation of cellular energy via the process known as oxidative phosphorylation. The enzyme contains two Fe and three Cu centers which together provide the redox machinery for the reduction of O2 to water. Recently, X-ray crystallography has provided the first three-dimensional description of the coordination spheres of the metal centers. However, the structures show the metal sites at low resolution, and in order to fully understand the mechanism of the reaction, it is desirable to determine the metrical details (bond lengths and angles) to much higher precision. X-ray absorption spectroscopy is unique in its ability to provide such detail, and we have applied the technique to determining the structure of the CuA center, a thiolate-bridged binuclear copper cluster in which the coppers are bridged by two cysteine ligands and have an extremely short Cu−Cu distance of ∼2.4 A. X-ray absorption spec...

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.

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.

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.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 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 CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is reported to be facilely inactivated by oxidant stress, To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD, In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 μM aerobic nitrofurantoin, or about 5 μM aerobic paraquat (PQ) and in about 1 min at 10 μM aerobic PQ. Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity. The K m was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth, Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none. DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made, DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO, Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60%. DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD. Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein-cofactor interactions that are unique to the cytochrome a-binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.

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 ba(3)-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 O(2)-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 ba(3)-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 ba(3)-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

To study the ligation of the Cu[sub A] site of heme-copper terminal oxidases, we have performed ENDOR measurements at X-band (9-GHz) and 35-GHz microwave frequencies on the three titled enzymes. The 35-GHz measurements provide complete spectral separation of the [sup 1]H and [sup 14]N resonances and permit analysis of the field dependence of the [sup 14]N ENDOR for each enzyme. The results indicate that two nitrogenous ligands were quite unequal hyperfine couplings are ligated to Cu[sub A] in each of the enzymes studied. We have also examined cytochrome caa[sub 3] isolated from His Thermus cells grown in the presence of D,L,-[[delta],[epsilon]-[sup 15]N[sub 2]]histidine. The 35-GHz Cu[sub A] ENDOR spectrum of this protein includes [sup 15]N ENDOR resonances whose frequencies confirm the presence of two nitrogeneous ligands; comparison with the [sup 14]N ENDOR spectra further shows that the ligand with the larger hyperfine coupling (N1) displays well-resolved [sup 14]N quadrupole splitting. The theory for simulating frozen-solution ENDOR spectra as refined here permits a determination of both hyperfine and quadrupole tensors for N1 of all three enzymes. These indicate that the bonding parameters and geometry of Cu[sub A] are well conserved. 55 refs., 7 figs., 1 tab.

Previous work (Dunham, W.R., Hagen, W.R., Fee, J. A., Sands, R.H., Dunbar, J.B., Humblet, C. (1991) An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions, Biochimica Biophysica Acta 1079, 253–262) suggested that under specific solution conditions and slow freezing times, samples of oxidized Chromatium vinosum (Cv) high-potential, iron-sulfur protein (HiPIP) form dimeric structures that exhibit characteristic spin-spin interaction in the EPR spectrum. In that study, it was also shown that two HiPIP molecules could approach each other along their Fe1-S4 axes to a distance of ∼ 13–14 A , as required by an analysis of the spin-spin physics. This is made possible because of a flattened surface on one side of the molecule within which S4 may, depending on side-chain motions, interact with solvent (Carter, C.W., Jr., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adman, E.T., Jensen, L.H. (1972) A comparison of Fe4S4 clusters in high potential iron protein and in ferredoxin, Proc. Natl. Acad. Sci. USA 69, 3527-3529). Here we describe a computer generated, hypothetical model of this proposed dimeric structure which suggests an energetically favorable interaction between two Cv HiPIP molecules and could account for the experimental observations. Two Cv HiPIP molecules brought together along their Fe1-S4 axes and maintained at a center-to-center distance of 14 A can be rotated with respect to each other so as to create complementary interactions between two glutamine residues, two phenylalanine residues, and two leucine residues, and an energetically unfavorable interaction between two arginine residues. Energy minimization calculations using the program XPLOR indicate that this arrangement may provide an overall energetically favorable interaction between the two HiPIP molecules that is strengthened by site-specific binding of Na and Cl ions.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, has been purified and extensively characterized as a two-subunit enzyme containing the metal centers characteristic of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c (Fee, J. A., Kuila, D., Mather, M. W., and Yoshida, T. (1986) Biochim. Biophys. Acta 853, 153-185). We have now cloned and sequenced the genes encoding the subunits of this enzyme. The smaller subunit consists of a typical oxidase subunit II sequence fused to a cytochrome c domain (Mather, M. W., Springer, P., and Fee, J. A. (1991) J. Biol. Chem. 266, 5025-5035). The larger subunit, the A-protein, is encoded by a fusion gene lying immediately downstream of the subunit IIc gene. The 5' portion of this gene encodes an oxidase subunit I homolog, whereas the 3' portion is homologous to oxidase subunits III. The A-protein from the purified enzyme appears too small from SDS-polyacrylamide gel electrophoresis and quantitative amino acid analyses to be a complete subunit I/III fusion, but it is currently not known if proteolytic processing occurs. Analyses of the sequences of oxidase subunits are presented which clearly identify T. thermophilus cytochrome caa3 as a bona fide member of the greater family of heme- and copper-requiring oxidases. As one consequence, it is confirmed that the set of invariant histidine residues (potential ligands of the metal centers) in cytochrome c oxidase subunits I and II is reduced to 8. Possible topological and helix packing models are developed based on considerations of homology, hydropathy, and variability.

The functioning of cytochrome

Cytochrome ba(3) from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169-16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba(3) and O(2) in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 --Ser variant in the ba(3) oxidase both reactions associated with proton uptake from solution, the P(R) --F and F --O transitions, were slowed compared to those of wild-type ba(3). The observed time constants were slowed approximately 3-fold (for P(R) --F, from 60 to approximately 170 mus in the wild type) and approximately 30-fold (for F --O, from 1.1 to approximately 40 ms). In the Thr-315 --Val variant, the F --O transition was approximately 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P(R) --F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical P(R) --F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O(2) reduction in the ba(3) oxidase.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdottir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.

While several Thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. We have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N(2)O. Previously, we characterized the heme a(3)-NO complex formed in cytochrome ba(3) from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu(B)(I) to form an end-on NO-Cu(B) or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo(3) from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N(2)O. The EPR and resonance Raman signatures of the heme o(3)-NO complex are comparable to those of the a(3)-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o(3)-NO complex does not produce a Cu(B)-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o(3)-NO.OC-Cu(B)] tertiary complex can form in bo(3) but not in ba(3). We assign these differences to a greater iron-copper distance in the reduced form of bo(3) compared to that of ba(3). Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu(B) is not an essential step of the reaction mechanism.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

As we in the field of rehabilitation begin to accelerate toward the twenty-first century, I believe we need to take a careful look at what awaits us on our doorstep. The evolution of technology that our forefathers witnessed in this century has made life far easier (and I believe better) for us than for any generation before us. As a member of the first generation of children to grow up in a special education setting (at least the first generation on Long Island, New York) I feel that I have witnessed an evolution of rehabilitation technology that is no less significant or astounding than that which my father and grandfather witnessed of technology in general. Just as this evolution of technology has greatly improved our lot on earth, so also will the application of this technology in every area of rehabilitation greatly improve the life of the disabled. In 1956, when I was in the first grade, the highest form of technology in our classroom was my electric typewriter. By the 1980s a microcomputer, such as Apple or IBM, was commonplace. It is my belief that the microcomputer will prove to be the most revolutionary device in the evolution of rehabilitation technology since the advent of the wheelchair! Indeed, no other device, current or conceptualized, has the potential to bring so much to the fingertips (no pun intended) ofthe disabled. One need only look at the proceedings from such conferences as RESNA and ICAART to recognize that almost all of rehabilitation engineering is bound to, or involved in, the computer in one way or another. There are several applications of this new computer technology that we can look forward to for further evolution. With this evolution will come the further enhancement of devices for those with disabilities. One of these areas is rehabilitation robotics. In this area of rehabilitation technology, the microcomputer provides the driving force and control behind a host of fascinating and useful devices.

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.