enzymesb - Enzyme mechanisms Biophysical Chemistry 1, Fall...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

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 Chemistr y 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Not immediately effective in delivering oxygen (lack of DPG) Potential and real shortages Stored blood quickly becomes outdated Foreseeable benefits of an oxygen carrier Emergency–trauma Cardiopulmonar y bypass surgery ANH: (extract blood before surgery, replace it afterwards)–red blood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 [Pα ] =k [P ][L] [Pα ] = k [L]; [Pβ ] = k [L] [Pαβ ] = k ⇒ [Pα β ] = k 2 [L]2 [Pα ][L] Note the the partition function is just the sum of the relative populations (concentrations) of all species: Q = 1 + 2k [L] + k 2 [L]2 = q0 + q1 λ + q2 λ 2 = N ∑ qi λ i i =0 More on allostery Now, compute the fraction of binding sites that contain ligands: y= ￿0￿ 2 1+ ￿1￿ 2 2k λ + 1 + 2k λ = ￿2￿ 2 + k 2λ 2 k 2λ 2 = 1 ∑ iqi λ i 2 ∑ qi λ i λ ∑ iqi λ i −1 λ ∂ ln Q = 2 ∑ qi λ i 2 ∂λ y= λ ∂ ln Q 1 ∂ ln Q = N ∂λ N ∂ ln λ Since this is uncoupled binding: Q = 1 + 2k λ + k 2 λ 2 = (1 + k λ )2 See Onufriev, Case, Ullmannn, Biochemistry 40, 3413 (2001) for a generalization. The Henderson-Hasselbach equation AH Q y =λ ⇔ A− + H + = 1 + kλ ∂ ln Q λk = ∂λ 1+λk Now, k = 10pKa and λ = 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 ] = κ ; µ ≡ [P ] [T ][P ] [R ] =L [T ] λ T TP R 0 1 1 2 1 kλ kλ k 2λ 2 µκ µκkλ µκkλ µ κ k 2λ 2 L Lck λ Lck λ Lc 2 k 2 λ 2 Q = (1 + k λ )2 (1 + µ κ ) + L(1 + ck λ )2 No phosphate y = = = = 1 ∂ ln Q 2 ∂ ln λ = λ 2Q ￿ ∂Q ∂λ ￿ ￿ λ ∂￿ (1 + k λ )2 + L(1 + ck λ )2 2Q ∂ λ λ [2(1 + k λ )k + 2L(1 + ck λ )ck ] 2Q (1 + k λ )k λ + L(1 + ck λ )ck λ (1 + k λ )2 + L(1 + ck λ )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 µ > 0, get a linkage between yL and yP . Linkage relations N yL = ∂ ln Q ∂ ln Q ; M yP = ∂ ln λ ∂ ln µ ∂ ln Q ∂ ln Q d ln λ + d ln µ ∂ ln λ ∂ ln µ = N yL d ln λ + M yP d ln µ d (ln Q ) = or (see pp. 25-26 in Slater): ￿ ∂ ln λ ∂ ln µ ￿ yP =− M N ￿ ∂ yP ∂ yL ￿ Let P = H + and L=O2 and N = 4: ￿ ∂ log[O2 ] ∂ pH ￿ yO 2 = M 4 ￿ ∂ yH + ∂ yO2 ￿ pH + + ￿ Hdeoxy − Hoxy 310, 9 1. Univ. 10, 1 . An example of some literature analysis Analysis of Cooperativity in Hemoglobin. Valency Hybrids, Oxidation, and Methemoglobin Replacement Reactions? A ttila S zabo a nd M artin K arplus* A BSTRACT: A n allosteric m odel proposed previously for s tructure-function relations in hemoglobin is applied. t o t he a nalysis of low- a nd high-spin valency hybrids. By a ssuming t hat t he low-spin oxidized c hains have t he tertiary s tructure of oxygenated c hains while t he high-spin oxidized c hains have a t ertiary s tructure i ntermediate between t hat of deoxygenated a nd oxygenated c hains, t he model p arameters a ss ociated with t he d ifferent valency h ybrids c an be o btained, To a nd t heir equilibrium properties c an be e stimated. T he hybrid results a re used also t o provide a n i nterpretation of methemoglobin a nd its ligand r eplacement reactions a nd of t he oxidation-reduction e quilibrium of normal hemoglobin. For t he various systems s tudied, it is found t hat t he effects of p H a nd 2,3-diphosphoglycerate a re i n a greement with t he model. u nderstand t he m echanism of cooperative ligand binding by t he hemoglobin t etramer, it is not s ufficient to know t he s tructure a nd p roperties of t he completely deoxygenated ( Hb) a nd fully oxygenated ( Hb(02)d) species. I nformation a bout t he i ntermediates ( Hb(O2), H b(02)2, Hb(02)3) that occur in t he c ourse of t he oxygenation r eaction is r equired. S uch knowledge is d ifficult to obtain in a highly cooperative system like hemoglobin because t he e quilibrium concentra- F rom t he lnstitut d e Biologie Physico-Chimique, U niversitt d e P aris VI, P aris 5e, F rance, t he M R C L aboratory of M olecular Biology, C ambridge C G 2 2 QH, E ngland, a nd t he L aboratoire d e C himie ThCorique, Universite d e P aris V II, P aris 5e, F rance. Received June 2 5, 1 974. S upported in p art by g rants f rom t he N ational Science Founda- tion ( GP36104X) a nd t he N ational Institutes of H ealth ( EY00062). A. S zabo was s upported by a fellowship from t he N ational Research Council of C anada. * Address correspondence to Department of Chemistry, Harvard University, C ambridge, Mass. 02138. BIOCHEMISTRY, VOL. 14, NO. 5 , 1975 931 odelshis tmathAyou’ve already seen! o xidation-reduction equic orresponding t reatment of t he T for he a nd B runori, l ibrium of n ormal hemoglobin is given in section 5. T he c oncluding discussion is p resented in section 6. of t hese a re ( I ) T hermodynamic D escription a nd t he A llosteric Model in which t he in s ome way. T he e quilibrium of a m acromolecule M with N b inding in which eis ites for a ligand X a t c oncentration ( activity) X c an be d en tonini a nd s cribed by a g enerating f unction ( Szabo and K arplus, 1972) e f errous ion d efined as: by c obaltous e t a l., 1 973; hich o ne t ype w here A , is t he m acroscopic equilibrium c onstant for bindo rphyrin I X ing of s l igands: odified por. , 1974), and M + SX S M X , ( 2) of e ither t he T he u tility of t he g enerating f unction, X ,y, lies in t he f act o rphyrin IX t hat e ach term A SAS,s = 0, 1, , . . N , is p roportional to t he a lysis of t he p robability t hat s l igands a re b ound. T hus, the f ractional n derstanding s aturation, ( y ) , of t he m acromolecule with l igand is given n g their poby: h e nature of h emoglobin. b rids, which t e p aper nil1 by t he s pin 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 l og L F IGURE 3 : Allosteric model for ligand r eplacement reactions; Hill coefficient n a nd scaled a ffinity log ~ 1 1 2 s. log L for c = 0.15, 0.20, v 0.25, a nd 0.30. s mall re We n to m eth which d (1973a) mental n ( see s more s t placeme meaning aquome tion in T his wo slightly m ental F or t he value of tivity ( n (see F ig Basic ideas of catalysis Carbonic anhydrase Enzymes N H Thr199 O O H O Glu106 O H C O His96 His94 N O O C H O O O Glu106 His119 His96 His94 H Thr199 O H C Zn2+ N H Thr199 161 O O H O C -O Glu106 Zn2+ His119 O H -N C His96 His94 S R O Zn2+ His119 FIGURE 5.2 The active site of α-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 involved in catalysis, whereas in class II it is generated directly on the catalytic subunit by the cleavage of adenosylcobalamin. The required electrons are also Ribonucleotide reductase provided from different sources. Class I requires oxygen for the generation of the NH2 N N O –O P O O P N O O P O O– H O– H OH O– N O H OH H RNR NH2 N N O –O P O O P N O O P O O– H O– H OH O– N O H H 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.) enhancesucleotide reductase Ribon the possibility for the nucleotide at the effector site to affect the nucleotide 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 tural Biology Ribonucleotide reductase et al. (2004) Structural mechanism of allosteric substrate specificity regulation in a ribo- Rnucleotide reductase. NreductaseBiol 11: 1142–1149. Copyright (2004) Nature.) ibonucleotide ature Struct Mol 439C—S 439C—S-H H C H—O H C O—H O C- O I E441 H H N I N437 H—O H O O CO I E441 H C- O I E441 H S—C462 O O—H H H N I N437 H—S—C225 H S—C462 439C—S-H H C H O H N I N437 C O H 439C—S-H H C H—O H O O S—C225 C- O I E441 S—C462 H H N I N437 C H—O O C- O I E441 H H C H O H N I N437 C O H H H—O O S—C225 C- O I E441 H S—C462 439C—S-H FIGURE 5.10 H C O H—S—C225 439C—S-H C C O H C O H H N I N437 O H H S—C225 H S—C462 439C—S H C O H H H—O S—C225 S—C462 O C- O I E441 H H C H O H N I N437 O H H S—C225 S—C462 The catalytic mechanism for ribonucleotide reductase (after Nordlund & Reichard, Mechanisms of serine protease hydrolysis Intermediates in serine proteases can be characterized letters a b c d Fig. 1 Hydrolysis of the acyl–enzyme 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 Å resolution, showing the acyl–enzyme 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 b Wilmouth et al., Nature Struct. Biol. 8, 689 (2001) c d Intermediates in serine proteases b c d Fig. 1 Hydrolysis of the acyl–enzyme 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 Å resolution, showing the acyl–enzyme 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 acyl–enzyme 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 Å resolution, showing the acyl–enzyme 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 Å). 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 Å 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 acyl–enzyme 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 Å 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 Å 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 acyl–enzyme 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 b d Fig. 2 Refined 2mFo acyl–enzyme 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 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 e 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 p calculated from the data to residues Ser 214, Phe 215 and Val 216 (Fig. 4). This motion is pH 10 (Fig. 1d) indicates visible but small (∼0.2–0.4 Å in the present structures) in the m the active site a side chain of Ser e. Electron denved for Wat 317 may reflect reenmately the same water molecule e elastase strucmolecule from ally observed in histidine is prode binding pocket wing the location l–enzyme complex pace filling model Asp 102 (brown) ptide complex at tetrahedral interghlights the active Both Wat 318 and tide are red in the ahedral intermedirmed by residues pocket) moves so ed by the peptide. that found in the b arbon olved g carell as d the them ion or valent e the ivated peraquires , even which en to long e not echmolxygen camphor according to the mechanism shown half-life of 10 min at 4°C in solution through the 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. autooxidation (9). Similar obstacles in Fig. 1. P450cam was the first member of Cytochrome P450 hydroxylates many substrates have pre- best which y, DeStrasse Group, ry, Los ed Biotaway, Fig. 1. Reaction pathway of P450cam. The catalytic cycle of Pseudomonas putida P450cam consists ossible. (It is this consideration that r use of the Laue method: The ion would reduce the P450.cam.O2 ile we were trying to observe it.) data set (s2) was collected after the same crystal for 3 hours with ngth x-rays to produce a larger hydrated electrons, thereby driving from the dioxygen species (5) toduced, activated oxygen intermedie third (s3) was collected after refreezing this crystal. This final ould—and indeed does— corree product complex (7). Specific each experiment are given in Tand the figure legends. All structures ined by molecular replacement nown structure of the oxidized 50cam complex (4 ) as a probe and as described (Tables 1 to 3). rting point: Structure of the fermphor complex (2). Apart from ges in the positions of some surand the presence of a bound tris he overall structure of the FeIIIcomplex (2) in the monoclinic ry similar to that determined by co-workers [Protein Data Bank e 2CPP] using an orthorhombic (4 ). The heme group is coached through its iron to the thiof Cys357. The heme is ruffled and ordinate iron atom is out of the lane by 0.3 Å. Camphor is orientdistal heme pocket by a single ond (2.9 Å) between its carbonyl m and the side-chain hydroxyl of Thr101 side chain is rotated com2CPP and forms a hydrogen bond the carboxylate of the propionic D pyrrole. The hydrogen bond hydroxyl group of Tyr 96 and the ygen of camphor shortened to 2.5 in 2CPP), resulting in a small thiolate ligand refined to 2.2 Å. However, the gen atom pointing toward Thr 252. Because (estimated distance error 0.2 Å). 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 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 A B C Schlichting et al., Science 287, 1615 (2000) Downloaded from www.sciencemag.org on November 11, 2009 2 normal Catalysis tance—substantially closerofthan Å—provided cumulate, so why mightrestraintsobservable by cytochrometheP450 here? Perhaps either the it be on P450 Fe–O 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- 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 ray–induced 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 Thr 252 in the reaction mechanism is to provide the P450.cam.O2 complex (6; Fig. 2C) that are a hydrogen bond. This is confirmed by the Catalysis by cytochromeisP450 not observed in (7theFig.possible P450.cam. in hydrostructures presented here: Thr oxyferryl structure ; 5C). Therefore, we 252 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 References and Notes 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 of Respiration, again Structural Biology Matrix H+ High pH II I H+ Intermembrane space ADP+ Pi –>ATP III IV H+ H+ Low pH 1 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 roton gradient across the membrane. This gradient drives the synthesis of AT ase. 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 II-Glu126 C O GO1 H O O H C N N H II O D372 H283 N III C HO HAS-C3D, A-ring N Fe O O54 H HAS-C2A, D-ring O H Cu H N H C O O H Y133 H O H O-H HH N H H O H282 N N H384 H HAS O11 H C 3 N H233 OH O Y237 H2O (O32) II-Glu15 O C OH O23 N HN O33 O N C H376 H NH H H R449 K-path + Hin H-O T302 The complete mechanism is probably very complex Thermodynamic analysis of this pathway of Respiration, again Structural Biology Matrix H+ High pH II I H+ Intermembrane space ADP+ Pi –>ATP III IV H+ H+ Low pH 1 In the respiratory chain, protons are pumped across the membrane by c dehydrogenase), III (cytochrome bc1 complex) and IV (cytochrome c oxidase roton gradient across the membrane. This gradient drives the synthesis of AT ase. 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 led δ in bacteria and chloroplasts. ledATP synthase at a cartoon ε in bacteria and chloroplasts. level OSCP F1 α β α F6 d Stator H+ H+ Rotor γ C10-15 a Fo b H+ H+ E 5.13 H+ H+ H + H+ Organization of mitochondrial ATP synthase. The proton gradient across the mem extended with His-tags and bound to a Ni-coated cover slip. The γ subunit was ATPattached to a fluorescently labeled actin filament. The actin filaments showed up synthase mechanism cartoon ADP+Pi βDP βE AT P* γ Pi P+ AD γ Pi P+ AD AT P i AD P+ P βTP γ βE βDP γ ATP γ βTP ATP ATP FIGURE 5.17 The hydrolytic mechanism of ATP synthase initially proposed by Boyer and subsequently modified according to crystallographic results. The three β subunits are shown in three different conformations during the functional cycle. The βΕ subunit is empty, βDP contains an already hydrolyzed ATP molecule, and βTP has an ATP molecule bound. When ATP binds to βΕ, the γ subunit rotates with respect to the β subunits. This leads to an activation of the ATP in the former βTP subunit. When the ADP and inorganic phosphate (Pi) is released, the continued rotation of the γ subunit leads to the open conformation βΕ. ATP synthesis proceeds in the opposite direction, driven by the proton gradient across the membrane. side. During a short passage when the rotating c subunit is close to the of the stator (Fig. 5.22), an arginine of the stator interacts with the glutamate ) that holds the sodium ion (proton). The ion is thereby released into the ATP synthase mechanism cartoon - H+D H+ aR277 +- H+ D- H+ D H+ a C10-15 DH+ DD- + H a D- + H D- + H H+ H+ H+ C10-15 D- + H D- + H 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 . 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 the ring of c subunits by a ratchet mechanism to a new position where they find lf 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 r (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 ...
View Full Document

This note was uploaded on 01/15/2012 for the course CHE 543 taught by Professor Staff during the Fall '10 term at Syracuse.

Ask a homework question - tutors are online