Expression, Purification And Measuring The Activity Of Cytochrome Oxi Dase C From A.ferrooxidans

Cytochrome c oxidase (common abbreviations: CO, CcO, COX or complex IV) belongs to the family of heme/copper enzymes. It is the terminal enzyme of electron transport chains in eukaryotes and some prokaryotes and as all its members COX, not only catalyzes the four-electron reduction of dioxygen to water, but also acts as a proton pump translocating protons vectorially across the inner mitochondrial mem-brane. In this redox reaction: O2(gas)+4CytC²⁺(aq)+4H3O+(aq)â†'4CytC3+(aq)+ 6H2O (aq), the energy is producted about 220 kJ/mol (at pH7). For each O2 molecule, eight protons are taken up from the inside, and four of these protons are pumped to the outside across the membrane. Cytochrome c oxidase is a multisubunit, which acts as a dimer, bigenomically encoded inner mitochondrial membrane protein. Eukaryotic COX is a complex enzyme, which consists of 11–13 subunits depending on the or-ganism. Prokaryotic homologues usually have a less complex organization, four sub-units as usual. However, subunit I and subunit II of various terminal oxidases show a high degree of sequence conservation, only both are essential for the function of the enzyme. This functional core catalyzes both oxygen reduction and proton pumping. Subunit II of cytochrome c oxidases contains the water-soluable binuclear Cu-Cu center: CuA center, which is the primary acceptor of electrons from reduced cytoch-rome c. Subunit I contains one low-spin heme A ("heme a") and a binuclear metal center formed by a high-spin heme A ("heme a3") and CuB. The electron-transfer pathway starts at CuA, which receives an electron from a cytochrome c on the outside of the membrane, from CuA the electron goes to heme a and then on to the binuclear center. In the catalytic redox action, the electron-transfer is coupled to proton-transfer, and the enegey, which is conserved by ATP, is required to remain nomal life activities.Acidithiobacillus ferrooxidans (A.ferrooxidans) is a chemolithotrophic bacterium which obtains energy through oxidation of ferrous ion to ferric ion with molecular oxygen as the terminal electron acceptor reduced to H2O. The Cytochrome c oxidase from A.ferrooxidans ATCC23270 is aa3-type enzyme. The core of the enzyme is formed by three subunits:CoxA,CoxB and CoxC, which are encoded by mitochon-drial(mt) DNA, the other subunit are encoded by the nuclear genome. The highly conserved CoxA is the biggest and most hydrophobic protein of the core subunits. It spans the inner mitochondrial membrane (IMM) with 12 transmembrane helices. There are three redox centers in the enzyme of CoxA: heme a3, CuB and heme a. CoxB contains CuA center. As mentioned above, we can conclued that CcO has four redox active center and CoxA plays a important in electron translation and proton-coupled- electron transfer.The CoxA gene of the cytochrome c oxidase from A.ferrooxidans was cloned through polymerase chain reaction amplification. The DNA fragments was detected and separated by agarose gel electrophoresis. After the extraction, purification and testing of the prokaryotic plasmid vector, the DNA fragments and the vector were di-gested by restriction endonuclease, separated by gel electrophoresis agarose and pu-rificated by special kits, respectively. We got restruction plasmid by connecting the DNA fragments with the vector under the condition of T4DNAigase, then successfully expressed in Escherichia coli(E.coli), finally purified the His-tagged CoxA by Nickel metal-affinity resin column chromatography to homogeneity. UV-vis scanning for the CoxA showed that there were two major absorption peaks and the reaction of CoxA with cytochrome c tested that CoxA plays a important catalytic role in the reduction of reduced cytochrome c. Through molecular structure modeling of cytochrome c oxi-dase, we can be more intuitive in understanding of its spatial structure, meanwhile it can be useful to analysis the structure of the catalytic active center.