Introduction to Heme Proteins Biomedical importance Structure of heme Structure and function of Mb Structure and function of Hb O 2 saturation curve of Mb and Hb differences The Bohr effect Effect of 2, 3 Bisphosphoglycerate Hemoglobinopathies – Met Hb – HbS – and thalassemias
Biomedical Importance Heme proteins function – O 2 binding – O 2 transport – Electron transport – Sickle cell disease: subunit of Hb altered. – Cyanide and CO poisoning – 2, 3 BPG (Bisphosphoglycerate) effect on Hb.
Heme Proteins Mb and Hb are heme proteins. Porphyrins are colored. – Iron porphyrin gives red color to blood. – Magnesium porphyrin gives green color to plants. Fe 2+ + protoporphyrin IX = HEME – Heme is a prosthetic group, or non-polypeptide unit necessary for the biological activity. – FerriMb- Iron is +3 oxidation state – OxyMb- Iron is +2 oxidation state and O 2 is bound to 6 th – DeoxyMb- Iron is +2 oxidation state
Heme Structure Four pyrolles linked methene bridges Four CH 3, 2 vinyl, and 2 propionate side chains could be arranged in fifteen different ways. Only one isomer, protoporphyrin IX, present. Fe 2+ Four nitrogen atoms Fe 2+ normally octahedrally coordinated. – 6 ligands – 4N of the porphyrin ring available – 2 remaining In cytochrome c 5 th and 6 th met and His In Mb and Hb 5 th coordinated to a His, 6 th is for O 2
Structure and Function of Myoglobin Myoglobin, a heme protein in heart and skeletal muscles. Myboglobin functions as – reservoir for O 2. – carrier for O 2. Myoglobin is single polypeptide chain (MW 17,000), 153 amino acids Surface is polar, interior is non-polar (characteristic pattern of globular proteins). 75% of polypeptide chain folded into eight stretches of -helical regions labeled A-H. Four segments terminated by proline.
Structure and Function Continued Heme group of Mb in crevice in proteins (between E and F helices). Cavity lined with non-polar amino acids, except two His residues. Proximal His binds Fe 2+. Distal His (E7) does not directly interact with heme group but helps stabilize ferrous form of iron. Therefore, Mb creates special environment for heme that allows reversible binding of oxygen without simultaneous oxidation of Fe 2+. His F8 means 8 th residue in F helix, it identifies it as a histidyl residue. F8 proximal His E7 distal His
More Structure and Function CO binds 25,000 times stronger than O 2 in isolated heme. Because no distal His in isolated heme. In biological systems, distal His forces CO to bind at an angle, decreasing affinity of CO to heme (200 times). This protects us against CO poisoning. Natural protection.
Electron-density Map of Myoglobin Near the Oxygen-binding Site
Benefit of Sigmoidal Curve Why Mb unsuitable as O 2 transport protein – PO 2 in lung 100 mmHg – PO 2 n venous blood 40 mmHg – It cannot serve as an effective vehicle for delivery of O 2 from lungs to periphery – After exercise PO 2 of muscle 5 mmHg. Then Mb releases its O 2 Sigmoidal curve allows Hb to deliver more O 2 to tissues in response to small S in PO 2
Salt links between and within subunits in deoxyhemoglobin
Hb has allosteric properties Allosterism (Allos: other, Steros: space): enzyme regulation that effector binds to one site on enzyme and increases or decreases the activity of another site.
Sickle Cell Anemia Genetically transmitted disease 4/1000 among blacks Sickle cell anemia homozygous Sickle cell trait is heterozygous HbS: Sickle cell Hb; HbA: Normal Hb – The only difference is substitution of Val for Glu at position six of the beta chains! Fetal DNA can be analyzed for sickle cell gene Val Glu results from T A mutation Detected by new techniques Sickle cell gene has missing cleavage site for restriction endonuclease which produces 1.3 kb fragment instead 1.1 kb fragment on gel electrophoresis DNA analysis performed early in pregnancy (8 weeks gestation)
Why HbS aggregates? DeoxyHbS – New hydrophobic patch Val6 interacts Phe85 and Val88 of beta chain of neighboring molecule to initiate aggregation. – Why aggregates do not form when HbS oxygenated In OxyHbS Phe85 and Val88 are largely buried inside Hb….