Oxygen reduction and proton translocation by cytochrome c oxidase

Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.Energian muuntaminen eliöissä on bioenergetiikan keskeisin dogmi. Energian otto on tehokkainta aerobisilla eliöillä verrattuna anaerobisiin eliöihin. Energian muuntamisen loppuvaihe tapahtuu aerobisten eliöiden mitokondrioiden sisäkalvolla hengitysketjussa tai anaerobisten bakteereiden solukalvolla. Hengitysketjun päätekompleksi on sytokromi c oksidaasi (CcO) entsyymi tämän tutkimuksen kohde. CcO toimii ensisijaisesti pelkistämällä happea vedeksi. Tämä tapahtuu kun CcO vastaanottaa kalvon toiselta puolelta elektroneja pieneltä ja liukoiselta sytokromi c entsyymiltä sekä protoneja toiselta puolelta. Tämän lisäksi CcO siirtää protoneja kalvon toiselle puolelle. Sekä hapen pelkistyminen että protonien siirtyminen vaikuttavat sähkökemiallisen kalvojännitteen syntymiseen jota solu käyttää ATP valmistuksessa ja erityyppisissä solun toiminnoissa. Vaikka CcO entsyymin rakenne tunnetaan aina 1,8 Å tarkkuuteen saakka, sen toimintaa ei tuskin pelkän rakenteen perusteella voi päätellä. CcO entsyymin sisäinen elektronin siirtoketju välivaiheineen tunnetaan kuitenkin hyvin. Toisaalta protonin siirtymisen perusteita entsyymissä on ennustettu kohdennetun mutageneesin avulla ja myöhemmin todistettu röntgensäde kristallografialla. Tästä huolimatta vankempia todisteita protonin siirtokoneiston yksittäisistä tekijöistä edelleen tarvitaan. Tässä työssä kehitimme uusia FTIR spektroskopiaan perustuvia menetelmiä CcO:n toiminnan selvittämiseksi. Näiden uusien menetelmien avulla pystyimme vastaamaan vuosia kiistanalaisina olleisiin kysymyksiin: [i] hapen kaksoissidoksen H+ donori on nyt tunnistettu sekä [ii] Glu-278, joka on protonin siirtomekanismin tärkeimpiä aminohappoja, proteolyyttiset siirtymät on nyt ensimmäistä kertaa osoitettu

Comparison Between O and OH Intermediates of Cytochrome c Oxidase Studied by FTIR Spectroscopy

Cytochrome c oxidase is terminal enzyme in the respiratory chain of mitochondria and many aerobic bacteria. It catalyzes reduction of oxygen to water. During its catalysis, CcO proceeds through several quite stable intermediates (R, A, PR/M, O/OH, E/EH). This work is concentrated on the elucidation of the differences between structures of oxidized intermediates O and OH in different CcO variants and at different pH values. Oxidized intermediates of wild type and mutated CcO from Paracoccus denitrificans were studied by means of static and time-resolved Fourier-transform infrared spectroscopy in acidic and alkaline conditions in the infrared region 1800–1000 cm−1. No reasonable differences were found between all variants in these conditions, and in this spectral region. This finding means that the binuclear center of oxygen reduction keeps a very similar structure and holds the same ligands in the studied conditions. The further investigation in search of differences should be performed in the 4000–2000 cm−1 IR region where water ligands absorb.Peer reviewe

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the “fast” kinetic spectra were found based on the following three requirements: (1) they are present in the “fast” phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the “slow” kinetic spectra in these mutants, and (3) they are absent in “fast” kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s)

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the “fast” kinetic spectra were found based on the following three requirements: (1) they are present in the “fast” phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the “slow” kinetic spectra in these mutants, and (3) they are absent in “fast” kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s)