Biophysics 490M Project

Transcription

1 Biophysics 490M Project Dan Han Department of Biochemistry

2 Structure Exploration of aa 3 -type Cytochrome c Oxidase from Rhodobacter sphaeroides I. Introduction: All organisms need energy to live. They degrade complex organic molecules found in their food to simpler products that have less energy. The most prevalent and efficient pathway to generate energy by degradation of complex molecules is through cellular respiration. In aerobic cellular respiration, oxygen is consumed as a reactant along with the organic fuel resulting in water as one of the products. Carbohydrates, fats, and proteins can all be processed and consumed as fuel in this process. Aerobic cellular respiration utilizes an electron transport chain to divide the energy release associated with combining electrons with oxygen into several energy-releasing steps instead of one large reaction in order to optimize the efficiency of converting the potential energy into useful work rather than being dissipated as heat. The high potential electron carrier NADH and FADH 2 from glycolysis and Krebs cycle are passed into the respiratory chain, consisting of several enzyme complexes embedded in the inner membrane of mitochondria or the cell membrane of bacteria. The final electron acceptor is O 2. As they catalyze different redox reactions for electron transfer, most of the enzyme complexes found in the respiratory chain form different organisms translocate protons across the membrane to generate proton gradient. The free energy released in the transfer of electrons along the chain is thus conserved in the form of a proton gradient, which can be used ATP synthase to synthesize ATP, the energy currency of the cell. Cytochrome c oxidase is the terminal enzyme of the respiratory chain in mitochondria and many bacteria. It catalyzes the reduction of oxygen to water and couples this redox reaction to the vectorial translocation of protons across the membrane. Shown below is the overall reaction this enzyme catalyzes: 4 cytc 2+ + O H H + in 4 cytc H 2 O + 4 H + out Rhodobacter sphaeroides aa 3 -type cytochrome c oxidase shares significant sequence and function similarities with mitochondrial aa 3 -type cytochrome c oxidase. It is a simplified version of mitochondrial enzyme, which makes it a very useful tool to study the structural and functional properties of mitochondrial enzyme. II. Structure review of R. sphaeroides aa 3 -type cytocrhome c oxidase 1. Overall structure Rhodobacter sphaeroides aa 3 -type cytochrome c oxidase has 4 subunits (Fig.1). Subunit I consists of 12 transmembrane helices, subunit II of two transmembrane helices and a

3 globular domain outside the membrane. These two subunits contain all the medal centers of this enzyme and are functionally important. Enzyme with only these two subunits is active, but its lifetime is only as much as 1% of that of wild type enzyme. Subunit III has 7 transmembrane helices which form a V-shape. It has been shown that the presence of subunit III prevents spontaneous inactivation of the enzyme by maintaining the structural integrity of the active center during oxygen reduction. Subunit IV has only one transmembrane helix. It has no direct interactions with any of the other subunits, and maintains its position by indirect contacts via some lipid molecules. Fig1. Overall structure of R.sphaeroides aa 3 -type cytochrome c oxidase (side view). blue, subunit I; red, subunit II; yellow, subunit III; purple, subunit IV.

4 A total of 218 water molecules per monomer were resolved in the R. sphaeroides enzyme structure. Although the majority of water molecules are found on the surfaces exposed to the solvent, many of them are also observed within the molecule; they form hydrogenbonded networks that are believed to facilitate proton translocation. 2. Metal center There are several metal ions and heme residues sitting in the enzyme, helping catalyze the redox reaction and translocate protons. The two coppers associated with subunit II form Cu A site. Electrons from cytochrome c are first transferred to the Cu A site, then to heme a, and then to the so-called binuclear center which is formed by heme a 3 and the nearby Cu B atom. Besides those redox-active metal centers, there are two non-redoxactive centers sitting in subunit I, Mg 2+ and Ca 2+. Mg 2+ might be involved in the transfer of proton in proton exit pathway, but the clear role of these two metal ions are not known. (a) (b) C A B Fig.2. Metal center. blue, subunit I; red, subunit II; yellow, subunit III; purple, subunit IV; cyan, Cu A and Cu B ; gray, Mg; pink, Ca; orange, heme a; green, heme a 3. (a). Overall structure of R. sphaeroides aa 3 -type cytochrome c oxidase (top view, only helices are shown). (b). Side view of metal centers. A top view of the enzyme in Fig.2. b shows the arrangement of all the helices and the positions of the metal ions. The helices of subunit I forms three semicircles. The semicircles together with the last segment of the previous semicircle have a pore-like appearance. The pores are blocked, pore A by mostly conserved aromatic residues, pore

5 B by heme a 3 and Cu B, and pore C by heme a and its hydroxyethylfarnesyl side chain. The two heme groups are sitting in the same level of the membrane (Fig. 2b). Their heme planes are perpendicular to the membrane, with an interplanar angle about 108 o. 3. Proton pathways: D& K pathway One important function of the enzyme is translocating protons from inside of the membrane to the out side of the membrane to generate proton chemical gradient. There are two proton transfer pathways in the enzyme (Fig. 3). One is called K-pathway because of a conserved lysine residue in this pathway, and the other is called D-pathway because it starts with a highly conserved aspartate residues. There are only 2 structurally resolved water molecules in the K-pathway, whereas 10 in the D-pathway. Those water molecules and the residues along each pathway might form hydrogen bonded network for proton transfer. out E286 CuB Heme a 3 S200 S201 Y288 K362 T359 N139 N207 S299 D132 D-pathway K-pathway Fig. 3. The D pathway & K pathway. Helices in blue contribute to D-pathway, and helices in yellow contribute to K-pathway. The K-pathway includes S (I-299), K (I-362), T (I-359), Y (I-288), etc. It is only involved in the first 1 or 2 protons transferred to the binuclear center for O 2 chemistry, while the

6 D-pathway is used to transfer both the chemical protons and vectorial protons. Starting from the aspartate 132 exposed on the protein surface, the hydrogen bonded network in D-pathway continues through N (I-139), N (I-207), S (I142), Y (I-33), S (I197), S (I-200), and S (I-201), up to the glutamate 286 near the binuclear center. The pathway beyond glutamate 286 is not clear. This glutamate has been shown to play a central role in controlling the proton-transfer kinetics during oxygen reduction. It has been proposed to be a switch point of chemical protons and vectorial protons, i.e., the proton from E286 is either transferred to the binuclear center for O 2 chemistry or to some unknown residues in the exit pathway, possibly the propionate of heme a 3, to be translocated out. 4. Proton pathway: the exit pathway Many water molecules are observed in the gap between subunits I and II in the crystal structure. These water molecules form a hydrogen bonded network and connect the propionate groups of heme a and heme a 3 to the outside of the molecule in many ways. E254 R482 Mg 2+ H411 R481 H334 Heme a 3 Cu B H333 Fig. 4. A possible proton exit pathway. The hydrogen bond network around one of the heme a3 propionate groups is shown.

7 Since it has been suggested that the propionate groups are involved in proton pumping, this network could be a possible proton exit pathway. Fig. 4 shows the hydrogen bonded network around one of the heme a 3 propionate group, which could be connected to E286 of the D-pathway in some intermediate states. The binding site of Mg 2+, consisted of E (II-254), H (I-411), and D (I-142), is part of this network. The importance of the Mg 2+ binding site is not clear since the enzyme is active without this metal binding site. 5. Structural comparison of WT enzyme and EQ (I-286) mutant enzyme As mentioned above, E (I-286) is proposed to play an important role in directing the protons from D-pathway to either the binuclear center or to the exit pathway. One possible way for E (I-286) to achieve this is by conformational changes of its side chain during the catalytic cycle. The alternative conformation may only exist transiently and therefore not be revealed in the static three-dimensional structure of WT enzyme. The EQ (I-286) mutation results in almost complete loss of oxygen reduction activity and inhibition of proton translocation. Study of the crystal structure of this mutant enzyme reveals that the mutation induces some conformational changes in nearby region whereas the structure of other parts of the enzyme is not affected (Fig.5). W(I-172) Heme a 3 M(I-107) Cu B H 2 O E/Q(I-286) Fig. 5 Superimposion of WT and EQ (I-286) mutant structures around E (I-286). Yellow, EQ (I-286); purple, WT enzyme.

8 In WT enzyme, the E (I-286) side-chain is hydrogen bonded to the carbonyl of M (I-107), however, in EQ (I-286) mutant, this hydrogen bond is disrupted. Additional conformational changes induced by the mutation include the rearrangement of the water molecules near this residue. What s more, a shift of M (I-107) and W (I-172) side chains and of a heme a 3 propionate group is observed. The comparison of the structures of the wild-type and EQ (I-286) mutant enzymes indicates that there is a correlation between protonation state and conformational changes of the E (I-286) side chain and structural changes around the heme propionates and other residues near E (I-286) position. In the D-pathway, there are other two mutations, ND (I-139) and ND (I-207), which result in decoupling feature of the enzyme, i.e., the mutant enzyme have wild-type oxygen reduction activity, but doesn t translocate protons. It is seemed that in these two mutant enzymes, the E (I-286) is locked in the position where only transfer of protons to the binuclear center is possible. If the crystal structures of these two mutant enzymes are available, the comparison of their structure near E (I-286) and that of wild-type enzyme may give more information about how the conformation of the side chain of E (I-286) move in the catalytic cycle. III. Summary In this paper, the general structure of the wild-type cytochrome c oxidase from Rhodobater sphaeroides is introduced. This enzyme has four subunits and five metal centers. Three of the metal centers, Cu A site, heme a, and binuclear center, are involved in electron transfer in this enzyme. Besides catalyzing the redox reaction, the enzyme acts as a proton pump, translocating protons from inside of the bacterial membrane to its periplasmic side. The two proton transferring pathways, D-pathway and K-pathway, are shown in detail. The role of the E (I-286) residue is discussed. The comparison of wild type and EQ (I-286) mutant enzyme structures shows the possible conformational changes different protonation states of E (I-286) may introduce. Based on the crystal structures, a possible proton exit pathway is proposed, which involves the heme a 3 propionate groups, Mg 2+ binding site and many water molecules between subunit I and subunit III.

9 Reference: 1. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Iwata, S. et al, Nature, 1995, 376: The X-ray structure of wild-type and EQ (I-286) mutant cytochrome c oxidase from Rhodobacter sphaeroides. Svensson-Ek, M. et al, J. Mol. Bio., 2002, 321: The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Tsukihara, T. et al, Science, 1996, 272: All figures in this paper are made by using program VMD.