How's the AA3 batch responding

Cytochrome c oxidase (CcO) is an integral membrane protein that is present in almost all aerobic organisms and forms complex IV of the aerobic respiratory chain. The redox energy of the oxygen reduction is used by the membrane-based complexes of the respiratory chain to transport protons across the membrane and thus to build up an electrochemical proton gradient. The complexes of the prokaryotic respiratory chain are located in the plasma membrane and the protons are pumped from the cytosol into the periplasm, while the eukaryotic respiratory chain is located in the inner membrane of the mitochondria, and the protons are pumped from the matrix into the intermembrane space. In addition to the actively pumped protons, other charges are shifted on both sides of the membrane: the electrons from reduced cytochrome cs are passed on from the outside of the membrane to the oxygen and the substrate protons for water formation get from the inside of the membrane to the oxygen, and these charge shifts and -neutralizations make up a total of 50% of the electrochemical potential. The stored energy of the produced electrochemical proton gradient is used by the adenosine-5´-triphosphate (ATP) synthase for the synthesis of ATP. The redox-active CcO has four metal centers that are involved in the redox reactions: the dinuclear CuA center, which receives electrons from reduced cytochrome c, the heme a and the binuclear heme a3-CuB center. In the binuclear Fea3-CuB center, the oxygen reduction to two water molecules takes place, in which four electrons and four substrate protons are required for each oxygen molecule. The catalytic cycle of CcO is divided into various intermediates that reflect the individual redox levels of the enzyme: the oxidized O state is converted to the E state by a one-electron reduction. A further reduction step leads to the two-electron reduced R state, which reacts to the P state through electron rearrangement after the binding of oxygen via the A state. The P state is named after a putative peroxy intermediate, but is actually a proven oxoferryl intermediate. In addition, a tyrosyl radical in the P state was identified by means of electron paramagnetic resonance (EPR) spectroscopy. The next reduction step, the reduction of the tyrosyl radical to the tyrosine residue, leads to the Oxoferryl intermediate F state. The fourth electron leads the CcO back to its ground state, the O state. Some of these catalytic states can be induced artificially by adding H2O2, whereby different intermediates are produced depending on the amount of H2O2 added. Equimolar amounts of H2O2 induce the PH state at high pH and the protonated F • H state at low pH. Both intermediates have the same number of electrons, i.e. the same redox state, but they differ in their degree of protonation. An excess of added H2O2 induces the FH state. H2O2-induced, artificial intermediates make it possible to study the catalytic cycle of the CcO in the model. In this work the type aa3 CcO of the soil bacterium Paracoccus denitrificans (strain ATCC 13543) was investigated. P. denitrificans CcO consists of four subunits compared to the bovine heart CcO, which is composed of 13 subunits. The crystallization of the functionally active two-subunit form of P. denitrificans CcO together with an antibody fragment (Fv fragment) enabled the structure to be determined at a resolution of 2.7 Å (Ostermeier, C., Harrenga, A., Ermler, U., Michel , H. (1997) "Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody Fv fragment." PNAS 94 (20): 10547-10553). 1) In this work a homologous expression system was used for the production of a recombinant wild type CcO. The gene of subunit I (ctaDIIbeta) of the CcO, which was expressed in the P. denitrificans AO1 deletion strain via the expression vector pUP39, has been cloned without affinity tags. The recombinant wild type CcO was purified in the same way as the native wild type CcO with the aid of the Fv fragment. The functional properties of the recombinantly produced wild type CcO were compared with those of the native wild type CcO. It could be shown that recombinantly produced wild type CcO compared to the native wild type CcO a) less activity b) shifts in the redox difference absorption spectrum c) low yield of UV-vis spectroscopically measurable intermediates (PH, F • H and FH states) and d) had less yield of EPR spectroscopically measurable tyrosyl radicals in the PH / F • H state. The observed differences could have been caused by the lack of redox-active metals in the recombinant wild-type CcO. The overexpression of the gene of subunit I of the recombinant wild-type CcO could have overwhelmed the machinery of protein synthesis and the recruitment of copper or heme group incorporation auxiliary proteins (heme / Cu chaperones). The investigation of the metal composition of the wild-type CcOs by means of total reflection X-ray fluorescence analysis revealed the correct ratio of redox-active metals of two Fe to three Cu for both proteins. For the bovine heart CcO it could be shown by another group of this research field that it selects mainly phosphatidylglycerol (PG) with (18: 1) -delta11-vaccenic acid residues from the stock of membrane lipids, although PG with (18: 1) -delta9- Oleic acid residues in the membrane occur much more frequently (Shinzawa-Itoh K. et al., (2007) Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase, EMBO, 26, 1713-1725). Due to its changed expression pattern, the AO1 deletion strain could have a different composition of lipids in its biomembrane, which could explain the reduced function of the recombinant wild-type CcO, since it could have bound non-functional lipids. ....