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Unformatted text preview: Enzyme mechanisms Biophysical Chemistry 1, Fall 2010 Hemoglobin and allosteric interactions Serine proteases Cytochrome P450 Cytochrome c oxidase Reading assignment: Chap. 5 Hemoglobin, blood and blood substitutes Chemistry and biochemistry of oxygen transport Practice and limitations of blood transfusions Two major ideas for red-blood-cell-free substitutes: Perfluorcarbon (PFC)-based ("un-natural"?) Hemoblobin-based ("natural") How these might be used, why progress has been so slow "IANAP" Overview of oxygen transport A tale of some very simple molecules... O2 : (di-)oxygen (20% of air) N2 : (di-)nitrogen (80% of air) CO: carbon monoxide (competes with O2 in hemoglobin) CO2 : carbon dioxide (by-product of respiration) NO: nitric oxide (control of blood pressure and vasoconstriction) C10 F18 : "FDC" (main component of fluosol) C8 F17 Br: "PFOB" (most promising perfluorocarbon) ... and a much more complex one Dangers and nuisances of conventional transfusions Dangers mild allergic reactions (1 in 30) acute respiratory distress (1 in 5000) hepatitis (1 in 30,000 to 100,000) [was 1 in 4 prior to 1965] HIV-AIDS (1 in 500,000) administrative errors (1 in 20,000) immune system suppresion Nuisances Blood must be screened and typed (ABO) Very limited shelf life (2-4 weeks) Not immediately effective in delivering oxygen (lack of DPG) Potential and real shortages Stored blood quickly becomes outdated Foreseeable benefits of an oxygen carrier Emergencytrauma Cardiopulmonary bypass surgery ANH: (extract blood before surgery, replace it afterwards)red blood cells not exposed to bypass circuitry and pump dissolution of air bubbles that lead to neurological dysfunction (50% incidence, severe incidence 6%) tissue and organ storage getting oxygen inside solid tumors simplified blood banking Perfluorocarbons Among the most inert materials known not immuogenic can be heat sterilized can be manufactured in large quantities Must be formulated into emulsions with surfactants most common surfactants are egg-yolk phospholipids oxygen solubility is 20 times greater than plasma extent of oxygen delivery is regulated by simply controlling pO2 PFCs are not metabolized, eventually excreted through the lungs with expired air A brief history of fluorocarbon emulsions 1966: Clark & Gollan show a mouse could live with oxygen-saturated PFC 1970s: Green Cross creates Fluosol, emulsion of FDC difficulty to formulate the emulsion excessively long retention times (2.5 years in animals) was used for a time for angioplasty, halted in 1994 2000s: Alliance and Baxter Healthcare create PFOB-based Oxygent now in phase II and phase III human trials Hemoglobin as a shape-shifter Regulation of affinity by phosphates DPG binds only in the bigger central cavity DPG binds only to the deoxy form of hemoglobin The deoxy form has lower affinity for oxygen DPG lowers oxygen affinity DPG is in red blood cells but not in the bloodstream itself a red-blood-cell free hemoglobin needs some other way to lower oxygen affinity "Mutant" hemoglobins offer ideas Main challenges for hemoglobin based substitutes protein itself too small and is rapidly excreted through the kidneys stabilize the tetramer form relative to dimers (e.g. by mutations) chemically cross link into oligomers oxygen affinity is too high in the absence of DPG search for modified hemoglobins with lower affinity (Hb Presbyterian) adverse vasoconstriction and blood pressure effects understand more fully how NO interacts with hemoglobin difficulty and expense of producing modified hemoglobins bacterial toxins contaminate proteins produced in E. coli expression in transgenic pigs, or in yeast, is still quite expensive Simple model of allostery [P ] = 1 [P ] =k [P ][L] [P ] = k [L]; [P ] = k [L] [P ] = k [P ] = k 2 [L]2 [P ][L] Note the the partition function is just the sum of the relative populations (concentrations) of all species: N Q = 1 + 2k [L] + k 2 [L]2 = q0 + q1 + q2 2 = i =0 qi i More on allostery Now, compute the fraction of binding sites that contain ligands: y= 0 2 1+ 1 2 2k + 1 + 2k 2 2 + k 2 2 k 2 2 = 1 iqi i 2 qi i = iqi i -1 ln Q = 2 qi i 2 ln Q 1 ln Q = N N ln y= Since this is uncoupled binding: Q = 1 + 2k + k 2 2 = (1 + k )2 See Onufriev, Case, Ullmannn, Biochemistry 40, 3413 (2001) for a generalization. The Henderson-Hasselbach equation AH Q A- + H + = 1 + k y = ln Q k = 1+k Now, k = 10pKa and = 10-pH ; hence: y= 10pKa -pH 1 + 10pKa -pH This yields the usual sigmoidal binding curve you learned about in high school. Hemoglobin-like model [T P ] = ; [P ] [T ][P ] [R ] =L [T ] 0 1 1 2 T 1 k k k 2 2 TP R L Lck Lck Lc 2 k 2 2 k k k 2 2 Q = (1 + k )2 (1 + ) + L(1 + ck )2 No phosphate y = = = = 1 ln Q 2 ln = 2Q Q (1 + k )2 + L(1 + ck )2 2Q [2(1 + k )k + 2L(1 + ck )ck ] 2Q (1 + k )k + L(1 + ck )ck (1 + k )2 + L(1 + ck )2 If L = 0, get simple non-cooperative binding; for L < 1 and c > 1 (that is, T state is favored in the absence of ligand, but the R state has a higher affinity), get "hemoglobin-like" cooperative binding. When > 0, get a linkage between yL and yP . Linkage relations ln Q ln Q ; MyP = ln ln NyL = d (ln Q ) = ln Q ln Q d ln + d ln ln ln = NyL d ln + MyP d ln or (see pp. 25-26 in Slater): ln ln =- yP M N yP yL Let P = H + and L=O2 and N = 4: log[O2 ] pH = yO2 M 4 yH + yO2 + + Hdeoxy - Hoxy pH An example of some literature analysis 310, 91. Univ. 10, 1. Analysis of Cooperativity in Hemoglobin. Valency Hybrids, Oxidation, and Methemoglobin Replacement Reactions? Attila Szabo and Martin Karplus* ABSTRACT: An allosteric model proposed previously for structure-function relations in hemoglobin is applied. to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied, it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model. understand the mechanism of cooperative ligand binding by the hemoglobin tetramer, it is not sufficient to know the structure and properties of the completely deoxygenated (Hb) and fully oxygenated (Hb(02)d) species. Information From the lnstitut de Biologie Physico-Chimique, Universitt de Paris VI, Paris 5e, France, the M R C Laboratory of Molecular Biology, Cambridge C G 2 2QH, England, and the Laboratoire de Chimie ThCorique, Universite de Paris VII, Paris 5e, France. Received June 25, 1974. Supported in part by grants from the National Science Founda- To about the intermediates (Hb(O2), Hb(02)2, Hb(02)3) that occur in the course of the oxygenation reaction is required. Such knowledge is difficult to obtain in a highly cooperative system like hemoglobin because the equilibrium concentration (GP36104X) and the National Institutes of Health (EY00062). A. Szabo was supported by a fellowship from the National Research Council of Canada. * Address correspondence to Department of Chemistry, Harvard University, Cambridge, Mass. 02138. BIOCHEMISTRY, VOL. 14, NO. 5 , 1975 931 models for the corresponding treatment of the This mathAyou've already seen! oxidation-reduction equiand Brunori, librium of normal hemoglobin is given in section 5. The concluding discussion is presented in section 6. l of these are ( I ) Thermodynamic Description and the Allosteric Model in which the in some way. The equilibrium of a macromolecule M with N binding s in which eisites for a ligand X a t concentration (activity) X can be deAntonini and scribed by a generating function (Szabo and Karplus, 1972) he ferrous ion defined as: by cobaltous i et al., 1973; which one type where A , is the macroscopic equilibrium constant for bindporphyrin IX ing of s ligands: modified porl., 1974), and M + SX S M X , ( 2) p of either the The utility of the generating function, X,y, lies in the fact porphyrin IX that each term ASAS,s = 0, 1, , . . N , is proportional to the nalysis of the probability that s ligands are bound. Thus, the fractional nderstanding saturation, ( y ) , of the macromolecule with ligand is given ing their poby: the nature of hemoglobin. ybrids, which te paper nil1 by the spin Plot curves as a function of parameters COOPERATIVITY IN H E M O G L O B I N c.020 12 c=O 1 5 c = o 20 c=025 0 -2 -I 0 I 2 3 4 5 log L FIGURE 3: Allosteric model for ligand replacement reactions; Hill coefficient n and scaled affinity log ~ 1 1 2 log L for c = 0.15, 0.20, vs. 0.25, and 0.30. small re We n to meth which d (1973a) mental n (see s more st placeme meaning aquome tion in This wo slightly mental For the value of tivity (n (see Fig Basic ideas of catalysis Carbonic anhydrase Enzymes 161 Thr199 O H O C Glu106 O H O His96 His94 Zn2+ N H O O O C N Thr199 O H H O His96 His94 Zn2+ H O O O C Thr199 O H H -N His96 His94 Zn2+ N H O S R O C O C -O Glu106 Glu106 His119 His119 His119 FIGURE 5.2 The active site of -carbonic anhydrase. Top left: The hydroxyl is ready for nucle- ophilic attack on the carbon dioxide. Top middle: The substrate HCO3 is bound with the protonated oxygen but not the negatively charged oxygen to the zinc ion. This is due to the fact that the OH group of Thr199 is hydrogen bonded to Glu106. The position and orientation of the subunit by the cleavage of adenosylcobalamin. The required electrons are also Ribonucleotide reductase involved in catalysis, whereas in class II it is generated directly on the catalytic provided from different sources. Class I requires oxygen for the generation of the NH2 N N O O O O P O O O P O H O H OH O H N N P O H OH RNR NH2 N N O O O O P O O O P O H O H OH O H H N N P O H FIGURE 5.3 The reaction catalyzed by ribonucleotide reductase (RNR). The 2' hydroxyl group of the ribose of ATP is replaced by a hydrogen atom to become dATP (Illustration kindly provided . by Derek Logan.) enhances the possibility for the nucleotide at the effector site to affect the nucleotide Ribonucleotide reductase binding to the active site. Loop 1 interacts solely with the nucleotide bound to the Active site Effector site Overall activity site Loop 2 Loop 2 FIGURE 5.6 Left: The organization of the dimer of the catalytic subunit (R1) of Class I RNR. The spatial relationship between the active site at the subunit boundary and the regulatory substrate specificity site is shown. The overall activity site is present only in some class I and class III enzymes. Right: Details of the binding site for effector (dTTP) and substrate (GDP). The illustration is kindly provided by Derek Logan. It should be noted that loop 2 is located between the nucleotide in the substrate specificity site and the one in the catalytic active site. Furthermore ctural Biology Ribonucleotide reductase RibonucleotideNature Struct Mol Biol 11: 11421149. Copyright (2004) Nature.) nucleotide reductase. reductase 439C--S H C H--O O C- O I E441 H H C O--H H--S--C225 H N I N437 H S--C462 O O 439C--S-H C H--O C- O I E441 H H C O--H H--S--C225 H N I N437 H S--C462 O et al. (2004) Structural mechanism of allosteric substrate specificity regulation in a ribo- 439C--S-H C H O C O I E441 H O H C H O H H N I N437 S--C225 O O H 439C--S-H C H--O C- O I E441 H H C H O H H N I N437 H S--C462 S--C225 O O 439C--S-H C H--O C- O I E441 H H C H O H H N I N437 H S--C462 S--C225 O S--C462 439C--S-H C H--O C- O I E441 O H H C H O H H N I N437 S--C225 S--C462 O O H 439C--S H C H--O C- O I E441 H H C H O H H N I N437 S--C225 S--C462 O H FIGURE 5.10 The catalytic mechanism for ribonucleotide reductase (after Nordlund & Reichard, Mechanisms of serine protease hydrolysis Intermediates in serine proteases can be characterized 2001 Nature Publishing Group http://structbio.nature.com letters a 2001 Nature Publishing Group http://structbio.nature.com b c d Fig. 1 Hydrolysis of the acylenzyme intermediate formed between -casomorphin-7 (YPFVEPI) and porcine pancreatic elastase. Scheme shows the deacylation reaction in which His 57 orients and deprotonates a nearby water molecule (Wat 317) to give a hydroxide that attacks the ester carbonyl, leading to the formation of the tetrahedral intermediate and subsequent product release. a, Refined 2mFo DFc electron density map at 1.6 resolution, showing the acylenzyme complex at pH 5 (data set 1, Table 1, see also http://www.ocms.ox.ac.uk/~rupert for atomic resolution structures). Intermediates in serine proteases letters a 2001 Nature Publishing Group http://structbio.nature.com b Wilmouth et al., Nature Struct. Biol. 8, 689 (2001) c d Intermediates in serine proteases b c d Fig. 1 Hydrolysis of the acylenzyme intermediate formed between -casomorphin-7 (YPFVEPI) and porcine pancreatic elastase. Scheme shows the deacylation reaction in which His 57 orients and deprotonates a nearby water molecule (Wat 317) to give a hydroxide that attacks the ester carbonyl, leading to the formation of the tetrahedral intermediate and subsequent product release. a, Refined 2mFo DFc electron density map at 1.6 resolution, showing the acylenzyme complex at pH 5 (data set 1, Table 1, see also http://www.ocms.ox.ac.uk/~rupert for atomic resolution structures). Intermediates in serine proteases d Fig. 1 Hydrolysis of the acylenzyme intermediate formed between -casomorphin-7 (YPFVEPI) and porcine pancreatic elastase. Scheme shows the deacylation reaction in which His 57 orients and deprotonates a nearby water molecule (Wat 317) to give a hydroxide that attacks the ester carbonyl, leading to the formation of the tetrahedral intermediate and subsequent product release. a, Refined 2mFo DFc electron density map at 1.6 resolution, showing the acylenzyme complex at pH 5 (data set 1, Table 1, see also http://www.ocms.ox.ac.uk/~rupert for atomic resolution structures). Ile 7 of the peptide is linked to Ser 195 in the active site, Glu 5 is present in two conformers and residues further away from the active site are disordered4. A water molecule (Wat 317) hydrogen-bonded to His 57 lies `poised' for catalytic attack onto the covalent ester bond (water to carbonyl carbon distance = 3.2 ). The carbonyl oxygen of the ester points into the oxyanion hole, forming hydrogen bonds with the backbone amides of Gly 193 and Ser 195. b, Difference Fourier map at 1.4 resolution, showing accummulation of the tetrahedral intermediate captured a minute after raising the pH of the crystal from pH 5 to pH 9 (data set 3, Table 1). Map calculated with coefficients Fo,tetra Fo,acyl and phases from the refined structure of the acylenzyme complex in (a). All significant features of the difference map were clustered in the active site and were recovered upon refinement in (c). c, Refined 2mFo DFc electron density map at 1.4 resolution for the assigned tetrahedral intermediate shown in (b). The tetrahedral oxyanion forms hydrogen bonds to the oxyanion hole and to a putative solvent molecule (Wat 318), which, in turn, forms a hydrogen bond with Thr 41. d, Refined 2mFo DFc electron density at 2.05 resolution, showing the active site 2 min after a jump to pH 10 (data set 4, Table 1). The peptide is released at this stage. A water molecule reappears in a position close to the position of the catalytic water (Wat 317) in (a). All refined electron density maps are contoured at 1.2 , where represents the root mean square (r.m.s.) electron density for the unit cell. Coloring scheme is defined as refined peptide = gold, and density covering the peptide and the two water molecules = blue. The difference Fourier map in (b) is contoured at half of the maximum peak height (4 ): positive density = blue, negative density = red and peptide = gray (unrefined). a buffer at raised pH (see Methods). At low pH, His 57 is protonated, and the attached water molecule4 (Wat 317 in Fig.1a) is a hydrogen bond acceptor from His 57. Raising the internal pH of the crystalline acylenzyme complex deprotonates His 57, changing the donor-acceptor relationship with Wat 317, thereby triggering deacylation (Fig. 1). After a given time at an elevated pH, each crystal was flash frozen in liquid nitro- gen. The experiments were repeated several times, and X-ray diffraction data recorded from a number of crystals (representative experiments shown in Fig. 1). Difference Fourier maps (for example, Fig. 1b), omit maps, the ARP/REFMAC6 procedure and SIGMAA-weighted refined 2mFo DFc electron density maps7 (Fig. 1) were used extensively in interpreting the structural data. Intermediates in serine proteases a c re Publishing Group http://structbio.nature.com Fig. 2 Refined 2mFo acylenzyme intermedia intermediate. The maps a resents the r.m.s. electron tide is colored gold. a, A perpendicular to the pla intermediate viewed fro c,d, The tetrahedral inte in (a) and (b). Nonbonde drawn as dotted lines. F (a,b), there is a possible h 317. In the tetrahedral in is possible between Wat oxygen. The dihedral an hedral intermediate are Ocarbonyl-C-OH = 99.1; O 113.5; C-C-OH = 117.8 denotes the oxygen of S the former carbonyl oxy complex and OH is from t b d ester at pH 5 (unpubli from http://www.ocms ric restraints were app structures. In the prese the tetrahedral interm Table 1) were refined restraints (the weights values used for conve compounds). This st adopt conformations c he main chain carbonyl of chain of Ser 214 links the potential motion of His 57 (ref. 10) Intermediates generated duringproteases the tetrahedral intermediate in serine the formation of ap calculated from the data to residues Ser 214, Phe 215 and Val 216 (Fig. 4). This motion is o pH 10 (Fig. 1d) indicates visible but small (0.20.4 in the present structures) in the om the active site a side chain of Ser me. Electron denrved for Wat 317 may reflect reenimately the same s water molecule ve elastase struce molecule from mally observed in e histidine is pro- ide binding pocket owing the location ylenzyme complex space filling model d Asp 102 (brown) eptide complex at e tetrahedral interighlights the active . Both Wat 318 and ptide are red in the rahedral intermediormed by residues g pocket) moves so ied by the peptide. that found in the b carbon volved ng carwell as nd the g them tion or ivalent ze the tivated mperaequires d, even which gen to s long ve not mechn moloxygen autooxidation (9). Similar obstacles in Fig. 1. P450cam was the first member of Cytochrome P450 hydroxylates many substrates have prethe P450 superfamily whose three-dimensional structure was determined, and both the vented direct observation of the remaining intermediates (5 and 7) by x-ray diffraction. e best which gy, DeStrasse Group, ory, Los ced Bioataway, Fig. 1. Reaction pathway of P450cam. The catalytic cycle of Pseudomonas putida P450cam consists Downloaded from www.sciencemag.org o camphor according to the mechanism shown half-life of 10 min at 4C in solution through possible. (It is this consideration that ur use of the Laue method: The tion would reduce the P450.cam.O2 hile we were trying to observe it.) d data set (s2) was collected after g the same crystal for 3 hours with ength x-rays to produce a larger hydrated electrons, thereby driving n from the dioxygen species (5) toeduced, activated oxygen intermedihe third (s3) was collected after d refreezing this crystal. This final hould--and indeed does-- correhe product complex (7). Specific each experiment are given in Taand the figure legends. All structures mined by molecular replacement known structure of the oxidized 450cam complex (4) as a probe and d as described (Tables 1 to 3). arting point: Structure of the feramphor complex (2). Apart from nges in the positions of some surand the presence of a bound tris the overall structure of the FeIIIcomplex (2) in the monoclinic ery similar to that determined by d co-workers [Protein Data Bank de 2CPP] using an orthorhombic m (4 ). The heme group is cotached through its iron to the thioof Cys357. The heme is ruffled and oordinate iron atom is out of the plane by 0.3 . Camphor is orientdistal heme pocket by a single bond (2.9 ) between its carbonyl om and the side-chain hydroxyl of Thr101 side chain is rotated com2CPP and forms a hydrogen bond the carboxylate of the propionic e D pyrrole. The hydrogen bond e hydroxyl group of Tyr 96 and the xygen of camphor shortened to 2.5 in 2CPP), resulting in a small thiolate ligand refined to 2.2 . However, the gen atom pointing toward Thr 252. Because 2 O binds more bent than CO (Table 4), the resolution of these crystal structures prevents a Again, intermediatesofcaninbe structurally characterized sterically induced displacement of camphor precise determination changes its length (estimated distance error 0.2 ). Again, no ordered water molecules are found near the vacant sixth position. upon ligand binding is smaller for O2 than for CO (6); nevertheless, some displacement is observed, providing additional evidence for the A B C Schlichting et al., Science 287, 1615 (2000) Downloaded from www.sciencemag.org on November 11, 2009 normal Catalysis tance--substantially closerofthan --provided cumulate, so why mightrestraintsobservable by cytochrometheP450 here? Perhaps either the it be on P450 FeO single-bond distance 1.8 the best fit to the observed electron density. The occupancy of the putative oxyferryl oxygen flexibility imposed by the crystal lattice or the unusual source of electrons, or both, com- A B Fig. 4. (A) Stereoview of comparison of the camphor complexes of ferrous (dark gray and dark blue water molecules) and ferrous dioxygen-bound (light gray and cyan water molecules) P450. Upon oxygen binding, camphor is displaced, two new water molecules bind, the backbone carbonyl group of Asp251 flips, and the backbone amide of Thr 252 rotates as does its side chain. (B) The interactions of the two new water molecules and the water chain extending from the first new water molecule 366 lytically treated crys intermediates we obse thawed the radiolytic about 30 s and collec (Tables 1 to 3). Thaw accompanied by no structure was determ placement. This exp with several crystals produces disorder. In tures show electron d the camphor C5 towar is still in the porphy consistent with the pr hydroxycamphor (4). similar to that deter co-workers from co-cr 5-exo-hydroxycampho includes a change i Asp251 carbonyl, whi the original positio P450.cam. We confir rayinduced generati camphor from frozen using gas chromatogr The P450 reaction ical route than two-ele binding. This is the p FeIII camphor compl oxygen intermediate can be generated by x so we cannot exclude product we observe ar The catalytic pat atomic resolution: mechanism. There i that these structures r a hydrogen bond. This is confirmed by the the Catalysis by cytochromeisP450 not observed in (7; Fig.possible P450.cam. in hydrostructures presented here: Thr oxyferryl structure 5C). Therefore, we 252 Thr 252 in the reaction mechanism is to provide the P450.cam.O2 complex (6; Fig. 2C) that are gen-bonding distance to the bound dioxygen interpret them as part of the proton delivery helix rearrangement, ther ing of a water molecule delivery path. (Indeed, b cules WAT901 and WAT the ferric complex, if on As required for this proton the side chain of Thr 252 r both the bound dioxygen water molecules WAT which could be the one protons for catalysis, as p inventory experiments (2 The structures presen vide valuable reference po reaction, either computat sign and synthesis of mo cause some of the most i drug metabolism and in involve hydroxylation o compounds, understandin has important practical co Note added in proof: have obtained evidence two electrophilic oxid course of P450 oxidati droperoxo-iron species oxo species. Partial occu oxo-iron species could b residual electron density of the putative activated and for elongated electr on the iron atom in the molecules in the asymm 1. For a comprehensive revie see P. R. Ortiz de Montella Structure, Mechanism an New York, ed. 2, 1995). 2. F. P. Guengerich, J. Biol. C References and Notes of Respiration, again Structural Biology Matrix High pH H+ ADP+ Pi >ATP I II III IV H+ Intermembrane space H+ Low pH H+ 11 In the respiratory chain, protons are pumped across the membrane by c Fee, Case, Noodleman, J. bc complex) and IV 15002 (2008) dehydrogenase), III (cytochrome Am. Chem. Soc. 130, (cytochrome c oxidase 1 proton gradient across the membrane. This gradient drives the synthesis of AT hase. Complex II (succinate dehydrogenase) does not pump protons. Cytochrome c oxidase shuttles electrons and protons Catalysis by cytochrome c oxidase Here are some key (proposed) intermediates 1 Fe-OH 7 Fe=O, Cu-OH Proton pumping pathways are partially known Hout O C O R449 O H + II-Glu126 H376 O O H GO1 H N O-H Y133 H H N H O H H O H C O H O C H H O54 O O H N HAS-C2A, D-ring HAS-C3D, A-ring H283 H Fe III O Cu H II H282 N N H233 H384 H HAS O11 H C 3 O H O Y237 H2O (O32) II-Glu15 O C O H K-path Hin + N N N N N N H C O33 N N H O C D372 H O23 H H H-O T302 The complete mechanism is probably very complex Thermodynamic analysis of this pathway of Respiration, again Structural Biology Matrix High pH H+ ADP+ Pi >ATP I II III IV H+ Intermembrane space H+ Low pH H+ 11 In the respiratory chain, protons are pumped across the membrane by c dehydrogenase), III (cytochrome bc1 complex) and IV (cytochrome c oxidase proton gradient across the membrane. This gradient drives the synthesis of AT hase. Complex II (succinate dehydrogenase) does not pump protons. ATP synthase at an atomic level A Textbook of Structural Biology FIGURE 5.12 Left: The complete ATP synthase molecule from mitochondria as seen by electron cryomicroscopy. The two large components of the enzyme, F1 and Fo, are primarily the top and bottom lumps of density. In addition to a central stalk there is also a thinner peripheral stalk lled in bacteria and chloroplasts. lledATP synthase at a cartoon in bacteria and chloroplasts. level OSCP F1 d F6 H+ H+ Stator Rotor C10-15 a H+ H+ H+ H + b Fo H+ H+ RE 5.13 Organization of mitochondrial ATP synthase. The proton gradient across the mem ATPattached to a fluorescently labeled actin filament. The actin filaments showed up synthase mechanism cartoon TP AD P+ P AT P* AT P i extended with His-tags and bound to a Ni-coated cover slip. The subunit was DP Pi P+ AD Pi P+ AD ADP+Pi E E DP ATP ATP TP ATP FIGURE 5.17 The hydrolytic mechanism of ATP synthase initially proposed by Boyer and subsequently modified according to crystallographic results. The three subunits are shown in three different conformations during the functional cycle. The subunit is empty, DP contains an already hydrolyzed ATP molecule, and TP has an ATP molecule bound. When ATP binds to , the subunit rotates with respect to the subunits. This leads to an activation of the ATP in the former TP subunit. When the ADP and inorganic phosphate (Pi) is released, the continued rotation of the subunit leads to the open conformation . ATP synthesis proceeds in the opposite direction, driven by the proton gradient across the membrane. n side. During a short passage when the rotating c subunit is close to the t of the stator (Fig. 5.22), an arginine of the stator interacts with the glutamate e) that holds the sodium ion (proton). The ion is thereby released into the ATP synthase mechanism cartoon H+D - H+ H+ DaR277 +D H+ H+ C10-15 Da D- + H D- + H D- + H a H+ DH+ C10-15 H+ H+ D- + H D- + H 1 The arrangement of the Fo unit by FIGURE 5.22subunits are made to rotate in the which the c The "push-and-pull" mechanism of ATP synthase. The aspartate s e. The a subunit forms half a channel forsubunits (blue) interacts temporarily with an arginine (aR277) of the a subu c the protons (or sodium ions) through which the aspartate on the c subunits where they bind. To escape, the protons (or sodium (a, orange). This shifts the balance in such a way that the aspartate loses its pro e the ring of c subunits by a ratchet mechanism to a new position where they find alf channel through the a subunit. Thisoutlet channel (upper) and the negatively charged form of the aspartate ca arrangement works like a water mill where the er (protons or sodium ions) drives the the arginine.to generate work. wheel around A proton will bind to the negatively charged aspartate of the next c the inlet channel (dim). Thus the excess of protons on the outer side of the mem "wheel" of c subunits and leads to the synthesis of ATP . outlet channel and a new ion is picked up at the inlet channel. The mec ...
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