Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba3 Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

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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 ...

Section: µSsupporting

confidence: 49%

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

Szundi

Funatogawa²,

Fee³

et al. 2010

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100

“…The results are consistent with the maintenance of the oxidized protein conformation (36), even though the metal centers are reduced, before annealing at warmer temperature allows the protein to change its structure. The strained configuration shows an interesting spectral feature at 589 nm, also observed in the bovine and thermus CcO irradiated crystals (34,37). Other spectral methods such as single-crystal low-temperature EPR or resonance Raman will be needed to determine the nature of this form; the Soret region of the spectrum, which could provide some clues, absorbs too strongly to be measured (Fig.…”

Section: S142mentioning

confidence: 91%

“…There is evidence that significant reduction of the metal centers occurs (34,35). Therefore, we measured the spectral characteristics of crystals of wild-type and mutant CcO at 100 K using the online microspectrophotometer available at BioCARS, Advanced Photon Source, Argonne National Labs (35).…”

Section: S142mentioning

confidence: 99%

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

Liu

Qin

Ferguson‐Miller

2011

Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362-and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.conformational gating | membrane protein structure | proton path mutants | X-ray reduction | water chain C ytochrome c oxidase (CcO) is a major contributor to and regulator of energy conservation in eukaryotic and many prokaryotic organisms. It uses four electrons from cytochrome c and four protons from the inner side of the membrane to reduce O 2 to water (1). In each catalytic cycle, four protons are also translocated across the membrane to generate a membrane potential, but the mechanistic details of coupling between proton pumping and the electron transfer events are still not fully understood (1, 2). Our recently solved crystal structures of the fully reduced form of Rhodobacter sphaeroides (Rs) CcO reveal conformational changes that were not previously observed (3), introducing mechanistic possibilities for coupling and gating of proton transfer, the significance of which we further elucidate in this report.Bacterial forms of CcO are similar in many respects to the mammalian mitochondrial CcO and their simpler subunit composition and ease of mutation have made them useful model systems for studying the mechanism of energy conservation, assuming it to be conserved. It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4, 5), and even those most closely related to eukaryotic oxidases (the aa ...

“…However, the structures obtained with conventional synchrotron light sources were determined from protein crystals exposed to a high X-ray flux and high levels of cryoprotectants at cryogenic temperatures. It has been shown in CcO (7,8) as well as other proteins and enzymes (9,10) that the high-intensity synchrotron X-ray beam has many deleterious effects including reduction, ligand dissociation, and damage (11,12). These deleterious effects cause irreversible changes in the protein structure leading to erroneous structural determinations, especially in protein systems containing electron-rich heavy metals (13).…”

mentioning

confidence: 99%

“…These deleterious effects cause irreversible changes in the protein structure leading to erroneous structural determinations, especially in protein systems containing electron-rich heavy metals (13). In oxidized forms of CcO, it has been shown that X-ray reduction of the metal centers occurs even with very short exposures to the X-ray beam (7,14). Deleterious radiation-induced artifacts are especially evident in the structure of the CO-bound derivatives (bCcO-CO) obtained at low temperature by synchrotron radiation in which the X-ray beam has been shown to photodissociate the CO from the heme a 3 iron atom (8).…”

mentioning

confidence: 99%

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Ishigami

Zatsepin

Hikita

et al. 2017

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.bioenergetics | X-ray free electron laser | crystallography | cytochrome c oxidase | serial femtosecond crystallography