2.  Prosthetic group with an iron atom in the middle of a porphyrin ring  Ring contains N, alkenes, and carboxylate groups  Commonly recognized as parts of hemoglobin (4 per)  Iron is responsible for binding oxygen in order to distribute to the rest of the body  Uptake of heme is one way the cell can bring in iron

3. http://omlc.ogi.edu/spectra/hemoglobin/hemestruct/ind ex.html Heme with iron atom bound in the middle of the porphyrin ring

4.  Intracellular ◦ Cytochromes (involved in cellular respiration) ◦ Proteins involved in DNA synthesis and cell division ◦ Extracellular  Hemoglobin  Myoglobin  Connective tissue, nervous system, immune system

5. http://sandwalk.blogspot.com/2007/08/heme- groups.html

6.  Soluble Fe3+ (ferric iron) – retrieved by compounds called siderophores  Ferriproteins  *Heme  Hemoproteins (Hb and Mb)  *Hemophores (proteins with high affinity for heme) *discussed in this presentation

7.  Entry into cells must be regulated because too much can be toxic  Requires a membrane protein because cannot naturally diffuse  Active transport mechanism ◦ Energy is derived from proton gradient ◦ Proton gradient formed and energy derived is transduced by proteins  Ton B or TonB – related proteins

8.  Escherichia Coli

9.  Carrier protein that brings heme to receptor  Serratia marcescens hemophore: HasA  188-residue protein  Very high affinity for heme  Beta sheet layer and 4 alpha helices  Heme iron is bound by coordination of His- 32 and Tyr-75 on opposing loops

10. http://www.pasteur.fr/recherche/unites/Mbbact/mbbact-en/research-01.html

11.  HasR  Can internalize both free heme or that bound to hemophore into periplasm  Has a weaker affinity for heme than HasA  Binds heme via 2 histidine residues  Uses energy derived from proton gradient to move heme to interior of cell

12.  HasA= hemophore (carrier protein that brings heme to receptor)  HasR = heme transport receptor  TonB/HasB = protein complex involved in transduction of energy from proton motive force  holoHasA = HasA with heme attached  apoHasA = HasA without heme

13.  HasA receives heme  Migrates to and docks onto receptor (HasR)  Heme transferred from hemophore to receptor  Heme passes into periplasmic space and enters cell  Hemophore HasA dissociates and can pick up more heme

14. http://www.pasteur.fr/r echerche/RAR/RAR2003 /Mbbact-en.html

15.  HasR can form tight complexes with both hemophores with heme (holoHasA) and those without (apoHasA)  Since HasA has high heme affinity, iron uptake can be very high at low [heme]  Good because too much reduced iron in the body is harmful

16.  When holoHasA is bound to HasR, heme is spontaneously transferred to receptor (no energy is required here)  Energy from proton motive force required for entry of heme into cell and apoHasA dissociation from HasR  HasB (paralog of TonB) transduces energy ◦ Signaling stimulus due to transcriptional autoregulation when HasA and heme bound to receptor

17. http://chemistry.gsu.edu/Dixon.php

18.  Determine function of entire heme transport system  2 ternary complexes: HasA-HasR-heme (WT and mutant HasR)  Binary complex: HasA-HasR  Resolutions: 2.7 angstroms for ternary complexes and 2.8-angstrom for binary complex

19.  WT ternary solved by MAD (multiwavelength anomalous diffraction)  Other two done by difference Fourier methods  Final residue counts: ◦ HasR= 752 residues ◦ HasA= 161 residues

20.  HasR contains 22 antiparallel beta-strands like other TonB-dependent receptors  Unlike others in the family, HasR has elongated extracellular loops (L2, L6, L9) – bind HasA  L7 and C apex used to attain heme

21. http://strucbio.biologi e.uni- konstanz.de/strucbio/ HasA-HasR-heme complex L6 L9 Heme (green) bound to L7

22.  Initially, heme-binding site of HasA oriented to face extracellular loops of HasR  Heme then binds to the two His residues of HasR (transferred about 9.2 angstroms) ◦ His-603 from L7 and His-189 from apex C of a plug that is common in these receptors ◦ Mutants of these two residues show no heme binding

23. http://ww w.pnas.org /content/1 06/4/1045.full Superposition of the heme groups attached to holoHasA(blue) and to HasA- HasR-heme (red)

24.  Spontaneous transfer from HasA to HasR  Transfer is endergonic (non-spont.)  Coupling of HasA and HasR is exergonic and exothermic (spontaneous)  Latter overrides former

25.  During complex formation, heme is not lost to solution  HasR-Ile-671 in L8 clashes with heme on holoHasA (Figure A)  Without the Ile, heme transport is not possible because the heme rotates to face HasA  Mutant with Glycine-671 used (Figure B)

26. http://www.pnas.org/content/106/4/1 045.full

27.  L7 and L8 of HasR displace the loop with HasA-His-32 causing break in coordination between residue and heme  Heme and HasA-Tyr-75 (stronger connection) still persists ◦ Stablized by deprotonation of phenol that H-bonds with HasA-His-83

28.  Later, the His-83 may get protonated and so the coordination is lost  Ile-671 displaces heme from HasA  Rotation of His-83 side chain prevents sliding back of heme to hemophore

29.  Shows that free heme can bind to HasR with apoHasA bound as well  There is a channel that goes from between loops 3 and 4 all the way to the heme binding site in which heme can travel

30. http://www.p nas.org/conte nt/106/4/10 45.full

31.  Mirrors ABC transport of cargo from bacterial periplasm to inside cell  Both have cargo molecule bound to protein that binds to and spontaneously transfers cargo to cis receptor  Energy is required (heme-proton motive; ABC-ATP hydrolysis) to get cargo to trans and dissociate protein from receptor

32.  How to get substance to protein with lower affinity?  Part of binding energy of donor to ligand is consumed when displacing the first loop (His-32)  Ligand transfer occurs when donor-acceptor come together due to steric clash (from Ile- 671)

33. Refinement -Resolution, Å 49.2–2.7 (2.73–2.70) 49.4–3.0 (3.03–3.0) 39.2–2.8 (2.83–2.80) -No. of reflections 99,334 (2,329) 77,295 (2,431) 92,482 (2,123) -Completeness, % 95.03 (71) 99.17 (93) 98.1 (71) -Rwork, % 23.7 (34.9) 21.4 (37.4) 22.6 (46.6) -Rfree*, % 27.3 (38.4) 24.3 (39.1) 26.2 (48.3) Model composition -Protein residues 1,850 1,850 1,850 -Heme atoms 86 0 86 -Water molecules 58 19 13 B-factors -Protein 93.5 80.2 110.6 -Heme 84.6 — 120.4 Deviation from ideal values -Bond lengths, Å 0.010 0.010 0.006 -Residues with bad bond lengths †, % 0 0.05 0 -Bond angles, ° 0.61 1.27 1.08 -Residues with bad bond angles †, % 0.22 0.71 0.550 Ramachandran plot † -Favored regions, % 92.4 89.6 89.7 -Allowed regions, % 99.2 99.5 99.1

34.  measure of how well refined structure predicts observed data  R-factors usually range from 0.2-0.6  Smaller R-factor is better  R-factors for the three structures are 0.237,.214,.226 for the WT ternary complex, mutant ternary, and binary complex, resp.  Shows well-defined structure