3/13/13 EVOLUTION OF HEMOCYANIN AND HEMOGLOBIN OXYGEN BINDING PROTEINS Lara Morrison, Parisa Monirzad, Lesley Crawford, and Robert Lim Background Respiratory proteins are not universal and differ among organisms both in color and chemical composition. Hemocyanin (Hc) Hemoglobin (Hb) http://www.lotusoverseas.com/human- circulatory-system-201.html http://www.fredmiranda.com/forum/topic/ 1181609 1) Molluscs 2) Arthropods 1) All invertebrates These blood proteins have evolved to efficiently transport oxygen in the circulatory system of various organisms with aerobic respiration. 1 3/13/13 Hemocyanin • Multi-subunit Metalloprotein with 2 copper binding sites • Cu-A site • Cu-B site • Less efficient at O2 transport than Hemoglobin which binds 4 O2. Hemocyanin Rx http://www.jbc.org/content/276/19/15563.long Oxygenation Blood= Blue Cu(II) Deoxygenated Blood= Colorless Cu(I) Mollusc vs. Arthropod Hemocyanin Molluscan Hemocyanin Arthropod Hemocyanin • 290,000 dalton molecule • 75,000 daltons molecule • One polypeptide binds • One polypeptide binds 2 O2 6-8 O2 molecules. molecules • Sequence may indicate it was • Cu-A and Cu-B site. duplicated 6 times within the 290,00 daltons. Ø Sequence analysis indicates Arthropod and Molluscan hemocyanin sequences only align at Cu-B site. Ø The two classes of hemocyanins share a copy of ancestral metal binding proteins, therefore evolution of Cu-B site is divergent evolution. Ø Large differences in a.a. composition and similarities in function between Cu-A sites indicates convergent evolution. Ø There was most likely duplication events of Cu-B site that lead to formation of Cu-A. 2 3/13/13 Evolution of Hemocyanin • Environment anaerobic in early evolution of life. • O2 produced by photosynthesis must have been toxic to organisms. • Enzymes evolved to neutralize O2 though oxidation reactions. • 2 metal ions used included • Copper (type 3 Cu) • Iron • O2 levels around 700-800 MYA closely represent those found presently. • Aerobic metabolism became established and body size increases with Cambrian Explosion. • A circulating oxygen transport protein became necessary to take advantage of aerobic metabolism. Ø Collabration work among Frederik Bang (top) and Jack Levin Which lead to discovery of blue blood during 1950’s Ø One crab died because of bacterial infection Ø caused almost the entire blood of the crab clotted Ø Only gram negative bacteria produce reaction 3 3/13/13 ? Ø Arthropods have Limulus amebocyte lystae (LAL) in blood cell Ø The white blood cells within the horseshoe crabs clotted when exposed to endotoxins. Ø Limulus – Taxonomic name of the horseshoe crab Ø Amebocyte – The white blood cells of the horseshoe crab Ø Lysate – Describes the original process used by Bang and Levin to rupture the cell membrane Why horseshoe crabs have the best immune system Ø horseshoe crabs have open circulatory system Ø LAL detect the presence of bacterial toxins Ø Formation of a gel-like clot to prevent bacteria from getting into their body http://www.horseshoecrab.org 4 3/13/13 Medicinal use of Blue blood Ø Blood is milky-blue due to the copper-pigmented hemocyanin molecule Ø Extract the blood and filtered to remove cellular debris Ø Less expensive and short process compare to any other animal Ø LAL is now used to test all drugs that are used intravenously such as vaccines. video Video • http://www.youtube.com/watch?v=e8KlAmtIu1E 5 3/13/13 Structure and Function • Hemoglobin (Hb) protein is arranged in a quaternary structure with 4 heme groups • Tetramer form = 2 α and 2 β subunits • Synthesized in the mitochondria in an immature RBC • Once matured, mammalian RBC’s do not have a nucleus • Whereas bird and most other species RBC’s are nucleated http://t1.gstatic.com/images Heme Group • Porphryin ring • Also known as a prosthetic group • Contains Iron ion (Fe2+) in the center • Cooperatively binds O2 • Conformational changes in Hb protein shape increase affinity to O2 http://www.ebi.ac.uk/ 6 3/13/13 Hb gene Switching from Fetus to Adult Embryonic Hb (HbE) – ζ2ε2, α2ε2 Fetal Hb (HbF) – α2Υ2 Adult Hb (HbA) – α2β2 W. G. Wood, Br. Med. Bull. (1976) 32:282-287. Evolution of Hemoglobin Mathews, van Holde, Ahern 3rd Edition 7 3/13/13 Lamprey as a Model Organism • First known organism to have Hemoglobin • Jawless, fish-like vertebrate • Considered the most primitive animal to posses • 500 million years ago • Dimer structure but not tetramer (α1β1) • Only weekly cooperative with O2 • First step in evolution of allosteric binding Long live the Wooly Mammoth ‘s HEMOGLOBIN • Reconstructed hemoglobin for the mammoth by using a bacteria reprogrammed with 43,000-year-old mammoth DNA • Although they are usually associated with the frozen north they originated from the tropics • Most artic animals arrange their blood vessels so that the arteries going down a leg can transfer heat to the veins coming up. The blood going to the toes is cold and animal conserves lots of heat from standing on the frozen ground 8 3/13/13 Problem…and solution! • Problem: red blood cells that takes on oxygen the lungs and delivers it in the tissues. The offloading process becomes much less efficient at low temperatures • The mammoth hemoglobin gets around this by lowering the amount of energy required to offload the oxygen. • Specifically, it uses tiny amounts of heat given off by a couple of reactions that are also underway on the hemoglobin molecule. • These other reactions relate to the binding of hemoglobin to other compounds like chloride and protons inside the red blood cells. The result is a far more energy efficient hemoglobin molecule. Animal without hemoglobin?!? • 80 years ago, rumored that there was a fish that had colorless blood. • Biologist disregarded it • Specimen was brought in and it contain almost all plasma. • Today, there are 15 known species of icefish of the family Channichthytidae 9 3/13/13 How is this possible? 1. Slow metabolism 2. The oxygen dissolves in water better at lower temperature then higher temperature What happened to the α and β? • β-globin completely missing from the genome, having been removed through a gene deletion mutation event • The gene encoding the α-globin has been deactivated and truncated 10 3/13/13 Discussion Questions 1. Given what we learned about the history of life in class, make sense of how both hemoglobin and hemocyanin evolved about 500 mya (hint Cambrian Explosion). References • Burmester, T. 2001. Molecular evolution of the arthropod hemocyanin superfamily. Mol. Biol. Evol. 18(2):184-195. • Hardison, R. 1999. The evolution of hemoglobin. Am. Scientist. 87(2): 126-135. • Honzatko, R. B. & Hendrickson, W. A. 1986. Molecular models for the putative dimer of sea lamprey hemoglobin. Proc. Natl. Acad. Sci. 83: 8487-8491. • N&am et al. 1997. The molecular evolution of arthropod & molluscan hemocyanin. Retrieved from http://www.nyu.edu/projects/fitch/resources/student_papers/nigam.pdf • Van Holde et al. 2001, Hemocyanins and invertebrate evolution. Jbc. Retrieved from http://www.jbc.org/content/276/19/15563.long • Weber, R. E., Behrens, J. W., Malte, H., & Fago, A. 2008. Thermodynamics of oxygenation-linked proton and lactate binding govern the temperature sensitivity of O2 binding in crustacean hemocyanin. J. Exp. Biol. 211:1057-1062. 11 3/13/13 Other Oxygen Carrier Proteins • Myoglobin – a single chain protein in mammalian muscle cells, increases ability to hold breath, found in high % in whales and seals • Cytoglobin – serves under hypoxia (not enough O2) • Neuroglobin – binds O2 with higher affinity than Hb in the brain to protect the brain • Leghemoglobin – found in plants such as soybeans • Hemerythrin – found in annelids and some marine invertebrates (bright pink) • Erythrocruorin – free floating, found in earthworms Wilber, A., Nienhuis, A. W., Persons, D. A. 2011. Blood. 117(15): 3945-3953. 12 .