True or false? Cyanide is poisonous because it binds more tightly to the iron in hemoglobin than does O 2 and cause suffocation. The Singapore government gets it right: “Blood Agents: Cyanide-containing compound, affect body functions by poisoning the enzymes, Cytochrome Oxidise. Hence preventing the normal utilization of oxygen by the cells and causing rapid damage to body tissues.” About Chemical Warfare Agents

False Let’s do the calculation. You have ~ 2g Fe in your body; about 1.5g of Fe is in your blood as hemoglobin. 1.5 g Fe = 0.03 mol. Toxicity of cyanide: The LD50 for ingestion is milligrams, or 1-3 milligrams per kilogram of body weight, calculated as hydrogen cyanide. The LC50 for gaseous hydrogen cyanide is parts per million. Inhalation of cyanide in this range results in death within minutes, with death coming more quickly as the concentration increases. Inhalation of 2,000 parts per million hydrogen cyanide causes death within one minute. So, if you weigh 60 kg, between mg could be a toxic dose. Or, g = mol CN- (as HCN) vs 0.03 mol Fe That amount of cyanide would block only ~10% of O 2 binding sites in hemoglobin, not enough to kill you. The toxicity of Cyanide is because of its strong binding to Fe in heme in other critical Heme-proteins.

The Many Role of Hemes oxygen carrier (hemoglobin) electron transfer (cytochromes a,b,c, etc, in respiratory chain) cytochrome oxidase (mitochondrial electron transport chain, oxygen is terminal electron acceptor and is reduced to water) detoxification (cytochrome P450, catalase) hydroxylation (cytochrome P450 in hormone production)

matrix cytosol Cyt c c1c1 c 2 x QH 2 QH 2 2 Q Q Complex III HEME 1 HEME 2 HEME 3 HEME 4

Fe(III)  Fe(II) Complex IV – Cytochrome C Oxidase Cu(II)  Cu(I) Cu(II)—Cu(I)  Cu(I)—Cu(I) HEME 2 HEME 3 HEME 4 HEME 1

Using Iron Porphyrins as Models for Hemoglobin The system:

Key Features of Hemes Fe oxidation state Fe spin state porphyrin oxidation state porphyrin hydrophobicity

Low Spin S = 1/2 n = 1 High Spin S = 5/2 n = 5 Intermediate Spin S = 3/2 n = 3 How will the spin state of Fe(porphyrin) complexes change on binding imidazole?

Sample for Evans’ Magnetic Susceptibility Method Inside capillary: sample in CHCl 3, 1) with imidazole 2) without imidazole Outside capillary: 99.5 %D CDCl 3 NMR tube

NMR Spectrum from Evans’ Method Inside capillary: sample in CHCl 3, produces broad singlet for paramagnetically shifted CHCl 3 below 7.3 ppm Outside capillary: 99.5 %D CDCl 3 produces usual sharp singlet for 0.5% CHCl 3 at 7.3 ppm 

Why is H resonance in CHCl 3 shifted downfield and broadened? pseudocontact and contact terms addition of new small magnetic field to local magnetic fields of neighboring nuclei is used in NMR Shift Reagents to “de-tangle” complicated spectra

How does shift, , relate to a magnetization of paramagnetic sample?  g = 3  0     c Mass susceptibility (+) Shift of signal, in Hz mass susceptibility of solvent -a diamagnetic contribution, a (-) value Magnetic field (400 MHz, or 400 x 10 6 Hz) Concentration of sample, in g/mL

Magnetic field lines of flux Magnetic field lines affected by a paramagnetic substance: attracts Susceptibility, X > 0 Magnetic field lines affected by a diamagnetic substance: repels Susceptibility, X < 0

How does mass susceptibility,  g, relate to unpaired electrons in a paramagnetic sample?  g x (Mol. Wt.) =  M  corr =  M -  diamagnetic corrections where  diamagnetic corrections for Fe, porphyrin, Cl, imidazole, a negative number!  eff = 3 R T  corr 1/2 = ( T  corr ) 1/2 N  2  eff = (n(n+2)) 1/2 Mass susceptibilityMolar susceptibility

Diamagnetic Corrections (cgs units) X o (CHCl 3 ) = x cgs Porphyrin: TPP= -700 x cgs TTP= -753 x cgs TClPP= -760 x cgs Fe = -13 x cgs Cl = -20 x cgs Imidazole = -38 x cgs

Low Spin S = 1/2 n = 1 High Spin S = 5/2 n = 5 Intermediate Spin S = 3/2 n = 3 2. How will the E red of Fe(porphyrin) complexes change on binding imidazole? 3. Will the E red potentials also reflect a change in spin state? 1. How will the E red of Fe(porphyrin) complexes vary with the porphyrin? Characterization by Cyclic Voltammetry

The Role of Axial Ligation and the Allosteric Effect in Hemoglobin O 2 Binding

3d orbitals on Fe Spin State of Fe affects size of ion

Large, high spin Fe(2+): In T state, transmitted by His on protein helix Small, low spin Fe(2+): In R state, transmitted to His on protein helix