Dataset Information

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus.

ABSTRACT: 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 ?M O(2) occurs with lifetimes of 9.3 and 110 ?s in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 ?s), 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 ?s 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.

SUBMITTER: Szundi I

PROVIDER: S-EPMC3000243 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus.

Szundi Istvan I Funatogawa Chie C Fee James A JA Soulimane Tewfik T Einarsdóttir Olöf O

Proceedings of the National Academy of Sciences of the United States of America 20101119 49

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 puls ...[more]

PMID: 21097703

Similar Datasets

Project description: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.

Project description:The Fea3(3+)···CuB(2+) dinuclear center (DNC) structure of the as-isolated oxidized ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba3 CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO's were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fea3(2+)···CuB(+) DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779-5783) revealed multiple (57)Fea3(2+) and (57)Fea3(3+) components. Moreover, one of the (57)Fea3(3+) components observed at 4.2 K transformed from a proposed "low-spin" state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin (57)Fea3(3+) component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the (57)Fea3(3+) components, and how the coordination and spin states of the Fea3(3+/2+) and Cu(2+/1+) sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba3 CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the CuB(+) site, and they may become potential proton loading sites during the catalytic cycle.

Dataset Information

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus.

Publications

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets