Reversible Oxygenation of Oxygen Transport Proteins

City University of New York (CUNY) CUNY Academic Works

Publications and Research Hunter College

1987

Reversible oxygenation of oxygen transport proteins

Charles Michael Drain CUNY Hunter College

Barry B. Cordon Tufts University

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This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] Reversible Oxygenation of Oxygen Transport Proteins

C. M. Drain and Bany B. Corden' Tufts University. Medford, MA 02155

A lecture demonstration illustrating changes in the visible Comparison of Oxygen Transport Proteins spectra of oxygen transport proteins upon reversihle oxygen binding is reported. The coloration upon exposure to oxygen Properly Hemoglobin Hemerythrin Hernocyanin is dramatic. Deoxyhemoglobin changes from purple to red, deoxyhemocyanin is transformed from colorless to blue, metal Fe Fe Cu while colorless deoxyhemerythrin turns red. This demon- metalldioxygen 111 211 211 metal coordination porphyrin protein protein stration illustrates an interesting example of a chemically number subunits 1,410 180 1, 2, 3, 4, 8 6, 12ta 120 reversible reaction and is used in the discussion of basic molecular weight 64,000. 108,0003 lo7= thermodynamic properties. A comparison of the physical Color characteristics of these oxveen storaee and transnort ~ro- oxygenated red burgundy blue . A teins is provided to illustrate the stru&ral diversity associ- deoxygenated purple colorless colorless ated with these proteins having a similar biological role. mL 02/100 mL blood 9-25 2 2-8 Organisms with a high surface-area-to-volume ratio oh- tain oxygen by diffusion;however, for large oreanisms diffusion is too inefficient. Coevolution of a &oxygen transport system to support respiration in the inner tissues is charac- teristic of higher organisms. Three distinct dioxygen transport and storage proteins have evolved: hemoglohin (Hb) and its monomer myoglobin (Mb), hemerythrin (HI), and hemocyanin (Hc). The dioxygen transport and storage proteins have different active sites as well as protein sequences that vary substantially between species. The prefix "heme" is derived from the Greek word for blood. Thus, confusion arises since the term "heme group"is associated with an iron porphyrin system, which actually is found in hemoglobin, but not in hemerythrin or hemocyanin. The active-site structure and dioxygen-binding geometry of hemoglobin (1-3), hemerythrin (4-7),and hemocyanin (8, 9) has been reviewed extensively. Significant investiga-

tive efforts indicate that there is a wide~ ranee~ n~ of~--~~~- active---.. fiite structures found in oxygen transport metalloproteins, as illustrated in the table and Fieure" 1. One of the most interesting aspects of these dioxygen transport proteins is that the geometrv of the dioxveen bond to the metalk). . differs in each case. There are tw~possihlemetal-dioxygen geometries in a 1:l complex, the end-on (10) eeometrv and theside- on (11) structure. F& 21 metal-dioiigeu complexes, there Figure 1. Active site shuctures far the oxygenated respiratory proteins: (a) hemoglobin and myoglobin. (b) hemerythrin. (c) hemocyanin. Histidine (H), ' Author to whom correspondence should be addressed. aspanic acid (D), glutamic acid (E).

Volume 64 Number 5 May 1987 441 are five possible bonding geometries, which are illustrated in There are no X-ray studies providing an uneauivocable Figure 2. The pressure required for one-half of the protein bondlength for the bound dioxygen in oiyhemoglohin; how- active sites to be coordinated PI,^) measures the binding ever, the bond angle is known for both oxymyoglobin (12) efficiency. Figure 3 illustrates the range of PI,%values for and oxvhemoelohin (13). These studies indicate the dioxv- each protein. gen is bound &d on as in Figure la. Much of our under&%- ine of hemoelobin comes from simnle model comnounds Comparison of Oxygen-Bindlng Proteins such as the picket fence porphyrin (I$, which exhibits ther- Myoglobin and its tetramer, hemoglobin, can be found in modynamic and s~ectrosconicproperties similar to the mvo- almost all vertebrates. some invertebrates. and even leeumes globin and hemoilohin active site;. and yeasts. Human myoglobin consists o€ a single poiypep- Hemerythrin is isolated from the erythrocytes in the coe- riderhain (elohin) of 150aminoacid residues.Theactivesite lomic fluid of sipunculids (spoonworms), priapulids contains one iron' atom coordinated by a porphyrin (heme) (tubeworms), brachiopods (lampshells), and polychaete an- with a hystidine in the axial position. The intense purple nelids (15). Hemerythrin is usually found as an octomer of color in the deoxygenated state is due to the conjugated molecular weight 108,000 that can be convertedto the mono- porphyrin system and is shifted to red when oxygenated. mer by chemical modification of the cysteine sulfhydryl group (16). Each subunit contains an active site with two Lon atoms coordinated directly to the protein amino acids that participate in hinding dioxygen. Upon hinding dioxygen, the colorless ferrous deoxyhemerythrin turns a bright salmon red. There are noX-ray structures of the oxygenated protein; however, resonance Raman studies indicate the dioxygen is hound to one iron atom in an end-on fashion (Fig. 2a) (17-19). X-ray crystallographic studies of azido- methemerythrin indicate the iron atoms are bridged by two amino acid carboxyl groups with an oxobridge between the iron atoms (Fig. lb). Hernocyanincan be found in the bloodof gastropods (aba- lone, whelk, snails, slugs), cephalopods (squids, octopi, cut- tlefish), crustaceans (lobsters, shrimp, crabs), arachnids (spiders, scorpions), and bivalve mollusks. Hemocyanin is an exceptionally large molecule with a molecular weight of up to 10"amu. Both comer. . atoms in the active site of hemocvanin are important in the hinding of dioxygen. In the oxygen- Figure 2. For proteins wllh two metal atom in the adive site, there anr five ated state theprotein is hlueand in thedeoxveenated stateit possible dioxygen binding geometries (a through e). Hemerymrin binds dioxy- -- gen in geometry (a). and hernocyanin binds dioxygen wilh geometry (c). is colorless. ~hestudy of several met (irreversibly oxidized proteins) forms (20-23) sunnort an active site eeometrv shown in Figure lc where the copper atoms in the &oxygenated protein are coordinated bv three histidine mouos-. and a (R group) from tyrosine. The copper atoms in the oxygenated protein are in a nearlv sauare pvramidal envi- ronment, and the roordination uf ihe &pe;atoms changes from four to five upon oxygenation. Resonance Raman (24) and electronic (23) studies indicate dioxveen." is bound bv both copper atoms'(see Fig. 2c). Cooperatlvny The most striking feature of hemoglobin is its cooperative binding of dioxveen.- Once a subunit of hemoelobin hinds an oxygen~molecule,it is thought the accompa&ng distortion of the active site causes potential energy to build in the protein. When a sufficient number of subunits bind dioxy- pen, the accumulated potential enerev... trieeers... a rotei in Eonformat~onchange. i'he remaining deoxygenatLd subunits exhibit a sianifirant increase in oxygen affinitv result- ing in a cooperative affect for dioxygen binding (2,251. The degree of cooperativity in hemoglobin depends on numerous factors, including pH, iunic strinpth, and the concentration of anions such as 2.3-diphosphoglyreraw, chloride, and ATP. Since the mechanism for coouerativitv in hemocvanin is thought to be significantly diffeient fro; thatfor &~o- glohin (8,26, 27), other factors are likely to be important. However, cooperativity is not yet demonstrated for hemerythrin and certain hemocyanin proteins.

Preparation ot Materials

General ~iguke3. Efficiencies of the respiratory protein compared by the range of All elassware and water used is sterilized. Caution should dioxygen pressure in which halt of the adive sites are oxygenated. P,,,. (Hb(T) be employed while handling the scalpel and broken whelk and HbiR) refer to hemwlobin before and aftw the Drmein conformation shells. Broken whelk shells are sham and the bacteria on change. Myoglobin is similar to HUT).) them can cause infection.

442 Journal of Chemical Education Isolation of the Bloods several months by storing the solutions in a freezer at -15 Hemoplobin from bovine blood is obtained from a veteri- OC. They will survive up to three freeze-thaw cycles. nary cliGc; 30 ml is filtered through a course glass frit and Conclusions diluted with 60 mL sterilized deionized water. Hemerythrin is obtained from the sipuuculid marine Since manv chemistrv students have bioloeical interests. a worm Phascolopsis gouldii2 by placing them in a tray and lecture demonstration emphasizing biologicky active corn- making an incision toward the center of the body and drain- pounds is appropriate. This demonstration can be su~nle- ing the coelomic fluid. The fluid from six worms, about 18 kented by dbser;ing the colormetric effects of an oxide& on mL, is filtered through a course glassfrit and diluted with 30 ferrous and cuprous ions. The cost of this demonstration is mL of sterilized, deionized water. less than $30, and some materials can be reused. Several Hemocyanin is obtained from American whelks Busycon experiments using hemoglobin and myoglobiu suitable for canaliculatum2 and is obtained by placing the whelk upside introductory, organic, or analytical chemistry lab courses down in a cool moist place for P6h to drain the sea water. have been developed (28-36). The whelk is bled by making a large incision parallel to the Literature Cned draining the ensuing into clean tray. foot and fluid a The foot 1. Senam, N. M.;Hunt,R.L. J. Cham.Educ. 1982,59,173. is the tough, cartilagelike part protruding from the shell. 2. Ochiai. E. Bioinorganic Chemistry:An Intraducfion: Allyn and Bamn: Boston, ,977: Three whelks provide about 36 mL of blood, which is subse- pp 103-124. 3. Callman, J. P. Aec. Chem. Re. 1977.10.265. quently diluted with 20 mL of sterilized, deionized water. 4. Kurfz,D. M.;Klofz,I.M. Ace, Chom.Rea. 1984.17.16. 5. Kurtz, 0. M.: Shriuer, D. F.: Klois, I. M. Cod. Chem. Rev. l977,24.145. Demonstration 6. Klofz,I.M.:Klippenstein.G. L.;Hendrickson, W.A.Scienca 1916,192,335. 7. Loehr. J.S.;Loehr,T.M. Adu.Inorg.Bimhem. 1979.1,235. The blood obtained is oxygenated. Completely deoxygen- 8. Senom,N.M. J. Chem.Edue. 1976,53,834. ating the protein is accomplished by the rapid bubbling of 9. Ref 2, pp 243-247. 10. Pading, L. Nofur8 (Londanl 1964.203.182. argon or nitrogen through the solution for 45 min. Avoid 11. Griffith. J. S. Proc.Roy. Soc., Ser A 1956,253.23, excess foaming of the blood solutions. Placing a hypodermic 12. Shasnan, B. Nature 1982,296,683, 13. Phi1lips.S.E. V.Nofure 1978,273,247. syringe needle at the bottom of the blood-water solution 14. Collman, J. P.:Ganpe. R. R.;Resd,C. A.:Halbert. T. R.:Lang,G.;Robinaon. W.T. J. contained in an Erlenmeyer flask fitted with a rubber sep- Am. Chem. Soc. 1975.97.1427. 15. Hyman, L. H. The Inuenabrafrs; McCraw-Hill: New York. 1959: pp 516-690. tum cap provides a satisfactory arrangement. The syringe 16. Klotl. I. M.; Kerosztes-Negy, S. Bioehemlalry 1963.2, MS. needle is attached to the gas supply by Tygon tubing. The 17. Kurtz, D. M.: Shriver, D. F.: Klofz, I. M. J. Am. Cham. Soc 1978.98,5033, container is vented by a short syringe needle with the tip 16. Du9L.L.:Klippansfoin,G.L.:Shriuer,D.F.:Klotz.I.M.Proc.Nof.Acod.Sei.,U.~A. 1961,78,4138. placed well above the solution. Oxygen or air is bubbled 19. (sl Shiemke,A. K.;Lahr,T. M.:Ssnders-hhr, J. J. Am. Chem. Sm. 1984,106,4951. through the solution to effect oxypenation. (h) Shicmke. A. K.: Loehr. T. M.; Sanders-hhr, J. J. Am. Chem. Soe. 1986,108. 2437. he color change associated kith oxygenation occurs 20. Co, M. 5.; Hodgson, K. 0.; Eceles, T. K.; Lontie, R. J. Am. Cham. Soe. 1981,103,9&1. within a minute. After noting the color changes, the re- 21. vanSehaik.E.M.:Schutter. W.P.;Gaykems,E.F.:vsnBruggen,E.J.;Hol, W.J. h Inuartrbrnles Oxygen Binding Prateina: Lsmy, J.: Lemy, J., Eda.: oekker: New versibility of dioxygen binding can be demonstrated during York, 1981: pp553-570. the course of the lecture by bubbling nitrogen or argon 22. Co, M. S.; Hdgson, K. 0.LAm. Chem. Soc. 1981,103.32W. throueh the solutions as described above. It takes about 15 23. Himmeiwright. R. S.; Eiekman, N. L.: LuBien, C.D.; Solomon.E. I. J.Am. Chom. Soe. " 1980,102,5378. min to deoxygenate these proteins to the point where reoxy- 24. Freedman, T. B.;Laehr, J. S.; Loehr, T. M. J. Am. Cham. Soc. 1976,96,28(19. eenation creates a noticeable color chanee. To illustrate the 25. Lahninger, A. L. P*imiples o1Biochamistry; Worth: New York, 1962: pp 169-202. 26. Senozan, N. M.; Landrum, J.: Bonavcntura, J.: Banaventura. C.; In Inuertebrofa differences, the UV-vis spectra of the &ree proteins in the Oxygen-Binding Ploteina; Lamy, J.; Lsmy, J.. Eds.: Dekker: Nm York, 1981: pp oxygenated and deoxygenated state can be presented. on=.""-..". ".* 27. Piekaft, S. M.; Ripps, A. F.;Larimer, J. L. Scionco I966,151,1W5. This procedure can be repeated many times before any 28. Warley, J. D.: Londo, T. J. Chem. Edue. 1983.60,650. loss of color for the oxy- and deoxy-forms due to denatur- 29. Heaney, A,; Wcller, D. L. J. Chrm. Educ. 1970.47.724. ation of the protein. The protein solutions can be saved for 30. Sawyer, W. H. J. Ch~m.Educ.1972.45.777. 31. Russo, S. F.; Sorstokke. R. B. J. Chem. Educ. 1973.50.347.

32. Solittncrber.A.G. J.~~~ Chem. -~Educ. 191C.51~~~. , 402 These animals are obtained from Marine Biological Labs, Woods $:I kplB".~.iJ.('hm Fdrr 1976.57 64 Hole, MA. Suitable animals may also be obtained from Pacific Bi* it ::::,>;: ~~~~d~.d&!~~~;$-' Marine Labs, Venice. CA. lo Br.~ta,nT. J Cnem E,IL,- 1962. 59, 2.X

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Volume 64 Number 5 May 1987 443