A novel cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochromeba3oxidase fromThermus thermophilus

Self Cite

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.

Section: Resultsmentioning

confidence: 99%

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba₃fromThermus thermophilus

Chang¹,

Choi

Vakkasoglu

et al. 2012

Self Cite

“…Atomic positions of amino acid side chains were taken from Protein Data Bank (PDB) code 1XME including also the topmost water molecule (43). The position of the bottom-most water molecule was taken from the structure of the E4Q II /K258R I mutant form of the enzyme, and the central water molecule (purple colored) was arbitrarily placed equidistant between the OG atoms of I-Ser-309 and I-Thr-312.…”

Section: Preparation Of Liposomes and Proton-pumping Measurementsmentioning

confidence: 99%

The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

Chang¹,

Hemp

Chen

et al. 2009

Self Cite

102

185

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 histidinetyrosine, 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.cytochrome c oxidase ͉ respiratory enzymes

“…The enzyme contains the four redox-active metal centers (8)(9)(10) and functions as a terminal oxidase for aerobic metabolism under limited oxygen concentration (8)(9)(10)(11). It also possesses NO reductase activity (12) suggesting shared evolutionary lineage of O 2 ∕NO reduction in this enzyme.…”

mentioning

confidence: 99%

CO impedes superfast O ₂ binding in ba ₃ cytochrome oxidase from Thermus thermophilus

Szundi

Funatogawa²,

Fee³

et al. 2010

Self Cite

100

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 doublelaser 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.double-laser technique | T. thermophilus ba3 | oxygen reduction | slow-fast kinetics | O2 channel T he reduction of dioxygen to water in the heme-copper oxidases takes place at the high-spin heme a 3 and Cu B heterodinuclear center (for review, see refs. 1 and 2). The reaction has been extensively investigated in several aa 3 -oxidases by timeresolved spectroscopic techniques in combination with the CO flow-flash technique (1, 2), in which the reaction is initiated by photolyzing CO bound to heme a 3 2þ in the presence of O 2 (3). The O 2 reduction has commonly been interpreted in terms of a unidirectional sequential mechanism (Scheme 1).The O 2 reduction in Thermus thermophilus ba 3 , a B-type oxidase with distant sequence homology to the A-type oxidases (4), has received much less attention (5-7). The enzyme contains the four redox-active metal centers (8-10) and functions as a terminal oxidase for aerobic metabolism under limited oxygen concentration (8-11). It also possesses NO reductase activity (12) suggesting shared evolutionary lineage of O 2 ∕NO reduction in this enzyme. In ba 3 , the thermal dissociation of CO from heme a 3 2þ in the dark is significantly faster (0.8 s −1 ) (5) than in the bovine aa 3 (0.023 s −1 ) (3, 13), and therefore CO flow-flash experiments on ba 3 require fast mixing; such experiments have recently been reported (6, 7). Moreover, the Cu B þ -CO complex formed following CO photolysis from heme a 3 2þ in ba 3 decays with a lifetime of approximately 30 ms (14), a rate much slower than that of O 2 binding to heme ...