Bio 98 - Lecture 7 Oxygen Binding Proteins Myoglobin and Hemoglobin

(Mb) (Hb)

Tetrapyrrole ring system Protophorphyrin IX Octahedral coordination

Heme biosynthesis

Octahedral coordination

Werewolves? Erythropoietic porphyrias are associated with accumulation of porphyrins in erythrocytes and are rare. The rarest is congenital erythropoetic porphyria (CEP) otherwise known as Gunther’s disease. The signs may present from birth and include severe photosensitivity, brown teeth that fluoresce in ultraviolet light due to deposition of type I porphyrins and later hypertrichosis. Hemolytic anemia usually develops. Recent use: the comic book Ultimate Comics Avengers Vol. 3 #1 (October 2010), in which writer Mark Millar employed porphyria as the explanation for vampires in that fictional universe.

C His64 His93 N

I. Similarities and differences • polypeptide length (~140 residues) & sequence (27 residues / 20% identical) are similar • presence of heme; mechanism of O2 binding Differences Mb Hb Location: muscle blood Subunits: 1 4 Function: O2 reserve O2 transport O2 source: blood lung O2 destination: mitochondria periphery O2 binding: simple complex hyperbolic sigmoidal (cooperativity)

A. O2 binding curve for myoglobin (Mb) II. O2 binding curves A. O2 binding curve for myoglobin (Mb) [O2] 0.5 1.0 [O2]0.5 Y (fraction of Mb with O2 bound, aka Mb:O2)

An equation of the form f(x) = x/(x + z) describes a hyperbola How does the availability of oxygen or [O2] influence the amount of Mb that has oxygen bound to it? Y = fraction of ligand binding site occupied (Mb:O2) [Mb:O2] bound Mb (1) Y = ——————— = ————— [Mb:O2] + [Mb] total Mb An equation of the form f(x) = x/(x + z) describes a hyperbola (2) binding rxn: Mb:O2 Mb + O2 [Mb] [O2] Kd = ————— = dissociation constant [Mb:O2] (just like the Ka of a weak acid)

Solve eq. 2 for [Mb:O2], then substitute into eq. 1 ... [Mb] [O2]/Kd [Mb] [O2] (1) Y = ————— = ————— [Mb] [O2]/Kd + [Mb] [Mb] (Kd + [O2]) [O2] (3) Y = ————— Kd + [O2] Another name for Kd is [O2]0.5 Mb:O2 Mb + O2 Kd = [O2] when Mb is half-saturated (Y = ½), i.e. when [Mb] = [Mb:O2]. At this condition, Kd = [O2], often written as [O2]0.5

Use pO2 (partial pressure) in place of [O2] (3) Y = ————— [O2]0.5 + [O2] Y pO2 Y = ————— P50 + pO2 Buffering of O2: Mb:O2 Mb + O2 blood mitochondria

B. O2 binding curve for hemoglobin Hb has a different function - transport, not buffering 1. Hb needs to pick up O2 efficiently in the lung where pO2 is high (100 mm Hg or Torr = 13.3 kPa). (1 Torr = 0.133 kPa) 2. Hb needs to drop off O2 efficiently in the tissues where pO2 is lower (30 mm Hg = 4 kPa). 3. Normal or “hyperbolic” binding won’t suffice; not enough difference in saturation when pO2 drops to only ~1/3 the maximal value (30 vs. 100 mm Hg).

p O2 [mm Hg] 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 q P50 = 2.8 mm Hg Simple (hyperbolic) myoglobin-like binding does not suit a transport protein well. pO2 in lung > < pO2 in tissues P50 = 26 mm Hg Y 8% 25% The state with a higher affinity for O2 has a lower P50 value (aka [O2]0.5) compared to the P50 value of the lower affinity state.

0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 q Sigmoidal (cooperative) binding does suit a transport protein well (Hb, magenta curve) pO2 in lung > < pO2 in tissues R-form 55 % Y T-form p O2 [mm Hg]

Change in conformation of heme as Hb goes from T -> R state His93 His93 His93 Val68 Val68 Val68 T (Tense): lower affinity for O2 (deoxy state) R (Relaxed): higher affinity for O2 (oxy state)

Change in conformation of subunits in tetramer as Hb goes from T -> R state Hb is an allosteric protein whose properties are affected by changes in quaternary structure, which are mediated by interactions with small molecules: i.e. O2 (and other effectors such as pH, see last two slides)

III. Models of Cooperativity A simple 2-subunit model 4-subunit models (Hb) Concerted model Sequential model

A simple 2-subunit model of cooperative ligand binding

B.1: Concerted model of the cooperative transition of Hb subunits: symmetry T (Tense): lower affinity for O2 (deoxy state) R (Relaxed): higher affinity for O2 (oxy state) Monod, Wyman, Changeux

transition of Hb subunits: induced fit B.2: Sequential model of the cooperative transition of Hb subunits: induced fit Some oxygen bound. Each binding of an oxygen molecule favors the transition of adjacent subunits to the strong-binding state and promotes their binding of oxygen T (Tense): lower affinity for O2 (deoxy state) R (Relaxed): higher affinity for O2 (oxy state) More oxygen bound. More and more subunits next to oxygen-occupied sites are switching to the strong-binding state. Koshland, Nemethy, Filmer

blood pH alters affinity of Hb for O2 in a useful way Bohr Effect blood pH alters affinity of Hb for O2 in a useful way tissue Y lung In the lungs, [CO2] is reduced (pH 7.6) compared to tissues (pH 7.2) 70 % 40 % CO2 + H2O H+ + HCO3- H+ and CO2 both bind to Hb and stabilize the T (deoxy) state: H+ and CO2 are allosteric effectors

BPG (2,3 bisphosphoglycerate) helps us adapt when climbing Mt. Whitney! BPG binds to positively charged groups stabilizing the T (deoxy) state. Increasing [BPG] lowers [O2] affinity, i.e. increases the P50. As you climb to higher altitude your body adapts to lower pO2 by increasing [BPG] from 5 to 8 mM. Y