Chapter I.1: Hemocyanin (Hc)

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1 Chapter I.1: Hemocyanin (Hc)

2 2 is essential for animals Low 2 solubility in aqueous solution (0.2 ml in 100 ml plasma) need some ways to carry 2 Exception otothenoids/family Channichthyidae Antarctic Icefish (body temperature: -1.7 o C) 2 absorbed and distributed directly by the plasma Can survive without 2 -carrier because of: u the high 2 content of the cold waters of the Southern cean u the good solubility of 2 due to the high pressure

3 Dioxygen reduction e - e -, 2 H + e -, H + e -, H H 2 2 H H 2 Thermodinamically unfavorable negative reduction potentiel Kinetically unfavorable spin state restriction H 2 E = V In ground state 2 is in triplet state ( 3 2 ) H 2 2 with 2 unpaired electrons in π 2p * orbital. Most of organic substances have a singlet ground states (no unpaired electron). ê unfavorable reactions E = V H H 2

4 espiratory metal-containing proteins Hemoglobin (Hb): Fe vertebrates (blood cells), echinoderms, annelids, molluscs, insect larvae Chlorocurorin: Fe (similar to myoglobin) annelids Hemerythrin (Hr): Fe marine invertebrates, annelids Hemocyanin (Hc): molluscs and arthropods

5 Dioxygen Carriers Properties Hemoglobin Hemerythrin Hemocyanin x. state in deoxy form (II) (II) (I) Metal n : 2 Fe: 2 Fe 2 : 2 2 : 2 Color deoxy form ed-purple Colorless Colorless Color oxy form ed Violet-pink Blue Metal coordination Porphyrin ring Prot. side chain (Glu, His) Prot. side chain (His) mol. weight (kd) to numb. subunits 4 8 many

6 Hemoglobin (Hb) Hemoglobin is an α 2 β 2 heterodimer α2 β1 β2 α1 β-chain

7 oxy-hemoglobin Fe III Back

8 Hemerythrin (Hr) His His His Fe II H FeII His His 2 His His His Fe III H His FeIII His 2 Back deoxy-hr oxy-hr

9 Hemocyanin (Hc) H H (I) (I) H H 2 H H (II) ( 2 ) (II) H H H H H H

10 Hemocyanin (Hc) Hc are respiratory proteins containing 2 atoms that reversibly bind a single 2. xygenation causes a color change between the colorless (I) deoxygenated form and the blue (II) oxygenated form. Hc carry 2 in the blood of most molluscs, and some arthropods. They are second only to Hb in biological popularity of use in 2 transport.

11 Hemocyanin (Hc). Hc is made of individual subunit proteins which contains 1 active site. Each subunit weighs about 75 kda. Subunits are arranged in chains or bundles in weights exceeding 1500 kda. Because of the large size of Hc, it is found free-floating in the blood, unlike Hb, which must be contained in cells because its small size would lead it to clog and damage blood filtering organs such as the kidneys. This freefloating nature allows for higher densities of Hc in the blood (as compared to Hb), and helps offset its low efficiency.

12 Crystal structure of hexameric Hc Panulirus interruptus (3.2 Å resolution) x 6 Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209:

13 90 Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209:

14 ligomeric forms of Hc 6 sub-u 12 sub-u 24 sub-u x 2 x 2 Decker H. et al. (2009) Structure 17(5):

15 Hc active site d- = 3.8 Å Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209:

16 xy form of Hemocyanin (oxy-hc) H H (I) (I) H H 2 H H (II) ( 2 ) (II) H H H H H H X-ray: d- = 3.8 Å EP: Silent EXAFS: d- = 3.67 Å aman: ν(-) = 749 cm -1 UV-vis.: 345 nm (ε = M -1 cm -1 ) 550 nm (ε = 1000 M -1 cm -1 ) EP: Silent

17 Coordination modes for 2 2- to a binuclear (II) center H H Terminal μ-η 1 (end-on) cis-μ-1,2 trans-μ-1,2 μ-η 1 :η 1 (end-on) μ-1,1

18 From enzyme to model and vice versa Metalloprotein H + 2 H + H 2 Biomimetic/bioinspired model H + 2 H + H 2

19 Biomimetic vs. bioinspired models rnithoptere (XVI e century) AirBus A380 (XXI e century)

20 meta-xylyl ligand Br H Br + H H K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372.

21 Terminal peroxo 2 EXAFS: d- = 3.67 Å aman: ν(-) = 749 cm -1 UV-vis.: 345 nm (ε = M -1 cm -1 ) 550 nm (ε = 1000 M -1 cm -1 ) EP: Silent k1 k-1 CH2Cl2 184 K k 1 > 10 6 M -1 s -1 EXAFS: d- = 3.31 Å UV-vis.: 385 nm (ε = M -1 cm -1 ) 505 nm (ε = 3000 M -1 cm -1 ) EP: Silent K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372; 1987, 109, 2668; 1988, 110, 6882.

22 I I 2 CH2Cl2 r. t. II II 1 e - reduction I II 2 CH2Cl2 r. t. II II K. D. Karlin et al. J. Am. Chem. Soc. 1992, 114, 7599; Inorg. Chem. 1992, 31, 3001.

23 tris-(2-pyridylmethyl)amine (TPA) H 2 + Br (ipr)2eth H H 2 + abh4 1 H 2 (ipr)2eth Br 3 3

24 μ-η 1 :η 1 peroxo (end-on) 2 k1 k-1 EtC or THF 183 K EXAFS: d- = 3.67 Å aman: ν(-) = 749 cm -1 UV-vis.: 345 nm (ε = M -1 cm -1 ) 550 nm (ε = 1000 M -1 cm -1 ) EP: Silent X-ray: d- = 4.36 Å UV-vis.: 525 nm (ε = M -1 cm -1 ) 590 nm (ε = 7600 M -1 cm -1 ) EP: Silent

25 Substituted TPA 2 k1 k-1 EtC 183 K = H & Me k 1 = 1.34/ M -1 s -1 k -1 = 2/ M -1 s -1 K = 0.67/ ΔH = -47.2/-49 kj mol -1 ΔS = -89/-87 J K -1 mol -1

26 Pyrazolylborates (scorpionates) H abh4 anisole - H 2 H B K

27 μ-η 2 :η 2 peroxo H B S 2 k1 k-1 EtC or THF 183 K H B B H EXAFS: d- = 3.67 Å aman: ν(-) = 749 cm -1 UV-vis.: 345 nm (ε = M -1 cm -1 ) 550 nm (ε = 1000 M -1 cm -1 ) EP: Silent X-ray: d- = 3.56 Å aman: ν(-) = 741 cm -1 UV-vis.: 349 nm (ε = M -1 cm -1 ) 551 nm (ε = 800 M -1 cm -1 ) EP: Silent Kitajima et al. J. Am. Chem. Soc. 1989, 111, 8975.

28 Structure of oxy-hc limulus polyphemus K. A. Magnus et al. Chem. ev. 1994, 94, 727.

29 2 k1 k-1 CH2Cl2 184 K unstable k 1 x 10 2 (M -1 s -1 ) k -1 x 10-5 (M -1 s -1 ) K x 10 7 H tbu F The life time of the dioxygen adduct is increased when is electron withdrawing group enabling characterization by resonance aman spectroscopy (only for = 2 ).

30 Biomimetic vs. bioinspired Hc models 2 CH2Cl2 184 K k 1 = M -1 s -1 Biomimetic 2 CH2Cl2 184 K k 1 > 10 6 M -1 s -1 Bioinspired

31 Spacer Spacer I I 2 II II 2 () n

32 k 1 = M -1 s -1 K = I I k 1 = M -1 s -1 K = CH2Cl2 184 K 2 ΔH = -58 kj mol -1 ΔS = -165 J K -1 mol -1 ΔH = -28 kj mol -1 ΔS = -101 J K -1 mol -1

33 T stable 2 : 2 adduct Kodera M. et al. (1999) J Am Chem Soc 121(47):

34 L(I) Spacer (I)L 2 k1 k-1 Spacer k1 Spacer L(II) (I)L L(II) (II)L 2 k-1 2 L(II) Spacer (II)L L(II) Spacer (II)L L(II) Spacer (II)L L(II) Spacer (II)L L(II) Spacer (II)L

35 1,4,7-Triazacyclononane (TAC) Ts H Ts Ts + Ts H Ts 1) CsC3/DMF 2) H2S4 3) X/Et3 S 2 k1 k-1 EtC or THF 183 K W. B. Tolman et al. Science 1996, 271, 1397.

36 Ph 2 () m II I () n CT II Absorbance d-d Ph () m () n λ (nm) () n ()m Ph n or m = 0 or 1 Eugénie Peyroux, Marseille Thesis 2008, December

37 L(I) L(I) 2 L(II) 2 k1 L(II) 2 (II)L k-1 fast rate determining step CH 3 II I ΔH = -0.7 kj mol -1 ΔS = -164 J K -1 mol -1 k 1 = M -1 s -1 k -1 = M -1 s -1 K = ΔH = -89 kj mol -1 ΔS = -240 J K -1 mol -1

Globins. The Backbone structure of Myoglobin 2. The Heme complex in myoglobin. Lecture 10/01/2009. Role of the Globin.

Globins. The Backbone structure of Myoglobin 2. The Heme complex in myoglobin. Lecture 10/01/2009. Role of the Globin. Globins Lecture 10/01/009 The Backbone structure of Myoglobin Myoglobin: 44 x 44 x 5 Å single subunit 153 amino acid residues 11 residues are in an a helix. Helices are named A, B, C, F. The heme pocket