Protein Function - Hemoglobin, Myoglobin Lectures 16-17

1 Protein function – hemoglobin, myoglobin Required reading: Chapter 6, pp. 265-289  2 REVIEW Proteins do most of the work in the cell. All proteins work by interacting with other molecules. REVIEW Proteins ar...

1 Protein function – hemoglobin, myoglobin Required reading: Chapter 6, pp. 265-289  2 REVIEW Proteins do most of the work in the cell. All proteins work by interacting with other molecules. REVIEW Proteins are essential for life and necessary for all cell processes, including DNA replication, RNA transcription, and protein translation  3 REVIEW The enzyme-substrate complex Active site: Region of an enzyme where substrate binds and catalysis takes place. Proteins fold to provide a framework (an environment) for the active site. Active sites contain residues required to active site bind substrate and also catalytic residues in residues that are directly involved in the lysozyme reaction mechanism chymotrypsin active site  4 REVIEW Many enzymes require additional chemical groups – cofactors Cofactors are non-protein compounds that are required for enzyme function. Can be simple inorganic metal ions or complex organic molecules called coenzymes Associate tightly or transiently ~30% of enzymes use cofactors Heme  5 REVIEW Cofactors, coenzymes, vitamins - bind to enzymes and aid in catalysis Coenzymes are small organic molecules that cannot by themselves catalyze a reaction but they help* enzymes to do so. Organic nonprotein molecules that bind to the protein (apoenzymes) to form the active enzyme (holoenzyme). Coenzymes cannot be synthesized by the cell - must be taken up in the diet – are often part of vitamins *provide a functional group that is involved in the catalytic reaction Prosthetic groups are coenzymes that are tightly bound; e.g., heme is a prosthetic group for hemoglobin.  6 Proteins Hemoglobin is a Hetero-tetramer Good example of protein Quaternary structure Myoglobin and hemoglobin (“globins”) 7 Critical proteins for our survival: oxygen transport and storage O2 is not very soluble in aqueous solutions like blood and can’t be transported freely to tissues – it also does not diffuse well across tissues – must be transported to the tissues, stored there until needed Myoglobin and hemoglobin are good examples of various levels of protein structure (as we discuss hemoglobin try to identify elements of secondary, tertiary, and quaternary structure) good example of various regulatory strategies, such as cooperativity (in oxygen binding) and allosteric control myoglobin (monomeric) hemoglobin (tetrameric)  8 Role of globins in oxygen transport & storage myoglobin and hemoglobin provide tissues with a continuous O2 supply hemoglobin is used for oxygen transport in all exhale inhale vertebrates and some invertebrates myoglobin is the oxygen storage protein used in all animal species hemoglobin also removes CO2 from tissues oxygen binding by globins is tightly regulated O2 is a “ligand” for hemoglobin and myoglobin  9 Myoglobin – oxygen storage Hemoglobin – oxygen transport present in tissues (e.g., muscles) present in blood monomer heterotetramer: 2 alpha (a), 2 beta (b) high affinity for O2 subunits (dimer of dimers) unaffected by pH, [CO2] or [2,3- moderate affinity for O2 bisphosphoglycerate] (BPG) sensitive to pH, [CO2] and [BPG] binds 1 O2 molecule binds 4 O2 molecules doesn’t bind BPG Myoglobin (Mb) 10 globular protein founds in muscles functions in oxygen storage heme 153 amino acids 77% a-helical eight a-helices: A, B, C, D, E, F, G, H - residues are often numbered within each helix - interior residues are non-polar except residue 7 of helix E (His E7) and His F8, which bind the heme group - exterior residues include both polar and non-polar amino acids binds to oxygen via a permanently bound cofactor/prosthetic group heme (aka haem) - heme consists of porphyrin and Fe2+ ion - heme binds O2 via the Fe2+ ion apomyoglobin (apoprotein): myoglobin without heme myoglobin: apomyoglobin + heme  11 Amino acid sequence alignments of globins Amino acid sequence alignments of globins. The dashes in the sequences indicate where theoretical gaps have been introduced to allow better alignments. The asterisk (*) indicates identical amino acids in all sequences, the colon (:) indicates a conservative substitution, and the period (.) indicates a semiconservative substitution. The eight α helices are outlined with red boxes, and the key His E7 and His F8 residues required for O2 binding are shown in blue. Structure of heme, the cofactor in hemoglobin and myoglobin 12 protoporphyrin IX heme pyrrole + + for the myoglobin-hemoglobin family, iron is chelated by a tetrapyrrole ring system called protoporphyrin IX heme = protoporphyrin IX + Fe2+ iron-storing transport molecules must be able to bind O2, not allow it to oxidize to any other substance, and release it on demand several enzymes are required to synthesize heme (what happens if heme is not properly synthesized?)  13 MEDICAL PROBLEM (with the answer) Are vampires real? Porphyria (a group of rare hereditary blood diseases due to a mutation in one of the genes that make heme) has been offered as a medical explanation for both vampires and werewolves. Porphyria characterized by the inability of the body to produce heme. Today there are effective treatments for porphyria. Without treatment, however, the porphyria sufferer must endure conditions such as extreme sensitivity to sunlight, excessive hairiness, skin sores, and disfigurement. In severe cases the fingers and nose sometimes fall off. The skin of the gums and lips might tighten and stretch, causing the teeth to appear very prominent and fang-like. Oddly enough, garlic, which stimulates heme production in healthy people, contains a chemical that worsens the painful symptoms of porphyria. Octahedral coordination of Fe2+ by heme, myoglobin and O2 14 The capacity of globins to bind oxygen depends on the presence of bound heme, which is responsible for the distinct red color of blood and muscles. The heme prosthetic group is wedged between the hydrophobic E and F a-helices. In addition to the 4 heme nitrogens, Fe2+ is coordinated by His F8 (for 8th residue on the F helix). Oxygen provides a 6th ligand and is stabilized by a hydrogen bond to His E7. The hydrophobic environment of the protein in the heme binding site keeps the iron in a reduced (Fe2+) form. His F8 (His93) His E7 (His64) * * G F8 E7 O2 (His93) (His64) E H F  15 When biochemists study how proteins bind to their ligands (L), they plot fractional saturations θ = occupied binding sites/total binding sites vs ligand concentration [L] The dissociation constant, Kd, can be determined from this plot by determining the concentration of ligand at which θ = 0.5  16 Both binding curves are hyperbolic curves and will approach θ = 1 at a high enough concentration of ligand. Example: Protein A has a higher affinity for ligand L than protein B does as indicated by the lower dissociation constant for protein A (10 nM) than for protein B (60 nM). Oxygen binding to myoglobin in tissues depends on the oxygen concentration 17 Binding curve for O2 binding to Mb + O2 MbO2 myoglobin (Mb). Oxygen concentration is measured as partial pressure (pO2). The fraction of myoglobin O2-binding sites occupied by O2 (q) is a function of the partial pressure of O2. Myoglobin binds tightly to O2: is 50% saturated at < 0.5 kPa. In resting cells (e.g., muscle cells) at ~4 kPa virtually all the myoglobin molecules (~95%) have O2 bound; i.e., myoglobin is saturated. (The plot is a saturation curve.) Note: pO2 in the lungs is 13- Metabolically active cells have a 14 kPa – much higher than very low pO2 (~ 1.3 kPa) causing O2 in the tissues – hemoglobin, not myoglobin, captures O2 to be released from myoglobin for from the lungs. use by mitochondria. q = 0.78. So, ~20% of the stored O2 is released from myoglobin in active cells – O2 is critical for muscle activity (respiration, oxidative phosphorylation, etc). Hemoglobin (Hb) 18 1 ml of human blood carries ~ 5x109 red blood cells (RBCs) each RBC contains ~3x108 hemoglobin molecules therefore, blood can carry ~2.5 mM O2, more than pure water (~1 mM O2) hemoglobin b2 b1 hemoglobin (Hb) has 4 subunits that are structurally homologous to myoglobin - 2 a subunits (141 amino acids each) tetramer - 2 b subunits (146 amino acids each) each a and b subunit individually looks like myoglobin, but only 27 amino acids (18%) are conserved (identical) among myoglobin and a and b subunits of Hb differences between Hb and Mb function are a2 a1 all due to the quaternary structure of Hb, and these differences are critical for Hb’s cooperative binding to O2 Hb’s allosteric regulation by CO2, H+, and BPG myoglobin 19  20 Structural changes of hemoglobin upon O2 binding binding of two oxygen molecules produces a significant change in the overall quaternary structure of hemoglobin and induces a T- to-R state transition - a “switch” the R (oxy) state has increased affinity for O2 - so binding of O2 at 2 subunits increases the affinity of the other subunits for O2 (as much as 100-fold!) - cooperativity the R state differs from the T state (Tense state, deoxy, low O2 affinity) by a rotation of about 15o of the a1b1 dimer with respect to a2b2 together with a shift that brings the b subunits closer together and narrows central cavity (as many at 50 non-covalent interactions between the two ab dimers are altered in the Tà R switch) (Relaxed state, oxy, high O2 affinity) Oxygen binding initiates structural changes 21 Structural changes in O2 hemoglobin are brought on “puckered” heme by O2-binding. Fe2+ Prior to binding O2 the Fe2+ His F8 is outside the plane of the heme and the heme is puckered F helix Upon binding of O2 the iron ion moves into the plane of the heme group, pulling with it His F8, which pulls ab interface F8 the F helix toward the heme a [The carboxyl terminal end of the F helix lies at the F helix F8 interface between the two ab dimers. Consequently, the structural transition at F helix the iron ion is directly b transmitted to the other subunits.]  22 Hemoglobin structure changes in the presence or absence of oxygen. Note the different positions of the F helix in the presence of O2.  23 REMEMBER: MYOGLOBIN IS A MONOMER its O2 binding curve is hyperbolic in shape  24 HEMOGLOBIN IS A TETRAMER its O2 binding curve is sigmoidal in shape due to COOPERATIVE BINDING  25 Hemoglobin binds O2 cooperatively and exhibits sigmoidal O2 binding curve This type of binding is very efficient since it permits full saturation of the protein in release saturation the lungs (or gills), where pO2 is high and efficient O2 release in tissues, where pO2 is low. Hemoglobin effectively has two states: deoxyhemoglobin (zero O2 bound) and oxyhemoglobin (4 O2 bound) It is either fully loaded or fully unloaded - not much of an intermediate. This “communication” between the different hemoglobin subunits regarding their oxygenation state (filled or empty) is only possible because of the quaternary structure of the protein. This cooperativity is not observed in myoglobin, which has a single subunit. Hemoglobin is optimized for oxygen transport; myoglobin for oxygen storage. Cooperativity between oxygen-binding sites in hemoglobin enhances 26 oxygen delivery hemoglobin must be able to bind oxygen under conditions of high pO2 (in lungs) and release it under low pO2 (in tissues) (= q) cooperativity means that hemoglobin has a higher affinity for O2 at high pO2 in the lungs and a lower affinity for O2 in the low pO2 of the tissues if O2-binding was not cooperative, O2 would either bind well at high pO2 but not release well at low pO2 ( ,