A “Sliding Scale Rule” for Selectivity among NO, CO, and O₂ by Heme Protein Sensors

Selectivity among NO, CO, and O₂ is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (<i>K</i>_D), the ratios for NO:CO:O₂ binding stay roughly the same, 1:∼10³:∼10⁶, when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a “sliding scale rule” for plots of log­(<i>K</i>_D) versus ligand type that allows predictions of <i>K</i>_D values if one or two are missing. The predicted <i>K</i>_D for binding of O₂ to <i>Ns</i> H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O₂ affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate, and imidazolate exert a “leveling” effect on ligand binding affinity. The reported picomolar <i>K</i>_D for binding of NO to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO <i>K</i>_D ratio of 1:∼10⁸. This deviation is explained by a complex, multistep process, in which an initial low-affinity hexacoordinate NO complex with a measured <i>K</i>_D of ≈54 nM, matching that predicted from the sliding scale rule, is formed initially and then is converted to a high-affinity pentacoordinate complex. This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O₂ binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation

Mechanism of Human Apohemoglobin Unfolding

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α₁β₁ interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α₁γ₁ interface is more resistant to dissociation than the α₁β₁ interface

Interfacial and distal-heme pocket mutations exhibit additive effects on the structure and function of hemoglobin.

Protein engineering strategies seek to develop a hemoglobin-based oxygen carrier with optimized functional properties, including (i) an appropriate O 2 affinity, (ii) high cooperativity, (iii) limited NO reactivity, and (iv) a diminished rate of auto-oxidation. The mutations alphaL29F, alphaL29W, alphaV96W and betaN108K individually impart some of these traits and in combinations produce hemoglobin molecules with interesting ligand-binding and allosteric properties. Studies of the ligand-binding properties and solution structures of single and multiple mutants have been performed. The aromatic side chains placed in the distal-heme pocket environment affect the intrinsic ligand-binding properties of the mutated subunit itself, beyond what can be explained by allostery, and these changes are accompanied by local structural perturbations. In contrast, hemoglobins with mutations in the alpha 1beta 1 and alpha 1beta 2 interfaces display functional properties of both "R"- and "T"-state tetramers because the equilibrium between quaternary states is altered. These mutations are accompanied by global structural perturbations, suggesting an indirect, allostery-driven cause for their effects. Combinations of the distal-heme pocket and interfacial mutations exhibit additive effects in both structural and functional properties, contribute to our understanding of allostery, and advance protein-engineering methods for manipulating the O 2 binding properties of the hemoglobin molecule.</p

Functional role of the 3₁₀-helix and adjacent residues: heme binding.

<p>(A) Ser-52, Ser-53, Arg-54, and Met-55 of IsdX1, designated SSRM, were each substituted to alanine and recombinant protein purified from <i>E. coli</i> as described in the <i>Experimental Procedures</i>. The absorbance properties immediately after purification from <i>E. coli</i> of wild-type (black) and SSRM (grey) IsdX1 were analyzed from 260–560 nm. (B) Recombinant IsdX1 was treated with low pH to remove co-purifying heme and the absorbance (250–500 nm) compared to the same preparation that was not acid treated. (C, D) Wild-type IsdX1, IsdX1-SSRM, or IsdX1 harboring mutations in Ser-52, Ser-53, Arg-54, or Met-55 were purified from <i>E. coli</i> and the heme content assessed by determining the ratio of the heme (399 nm) to protein (280 nm) absorbance (referred to as “bound heme”). In (C), the relative amount of associated heme is recorded following the purification of each IsdX1 variant from <i>E. coli</i>. In (D), all endogenous heme was removed from the preparations as described in (B) and apo-proteins incubated with 5 µM heme for 10 minutes at 25°C, followed by absorbance measurements. The absorbance value of a heme-only control (5 µM) was subtracted from all IsdX1 plus heme reaction readings. The values in (C) and (D) represent the mean and standard deviation of three independent experiments. The asterisk (*) means the differences were significant (p<0.05).</p

Rate constants for heme dissociation from IsdX1 variants.

a<p>The halftime is defined as the amount of minutes for one-half of the heme to dissociate from IsdX1.</p>b<p>For S53A and R54A, the dissociation curves are best described by two phases, each with a single rate constant. The percentages indicate the proportion of the total population giving that particular rate.</p

Heme dissociation kinetics for wild-type and mutant IsdX1.

<p>Wild-type or mutant (S52A, S53A, R54A, or M55A) IsdX1 were purified from <i>E. coli</i> and endogenous heme removed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002559#s4" target="_blank">Materials and Methods</a>. Proteins were re-constituted with heme, excess heme removed by gel filtration chromatography, and holo proteins (1 µM) mixed with H64Y/V68F apo-Mb (26 µM) at 25°C in PBS, pH 7.4. Time courses for hemin dissociation the IsdX1 variants were determined by measuring the difference between in the increase in absorbance at 419 nm (peak for holo H64Y/V68F holo-Mb) and the decrease in absorbance at 380 nm (strong absorbance by holo-IsdX1).</p

Functional role of the 3₁₀-helix and adjacent residues: hemophore and NEAT-domain biology.

<p>Purified wild-type or mutant IsdX1 were added to a final concentration of 1 µM to hemophore-deficient (Δ<i>isdX1</i>, Δ<i>isdX2</i>) <i>B. anthracis</i> Sterne 34F2 grown in iron-chelated RPMI with or without hemoglobin (10 µM) and the OD₆₀₀ recorded at 2, 4, 6, and 8 hours. The results represent the mean and standard deviation of three independent experiments. The asterisk (*) means the differences were significant (p<0.05). Hb = hemoglobin.</p

Superimposition of apo-IsdX1 and holo-IsdX1.

<p>(A) Ribbon representation of superimposition of apo-IsdX1 (pink) and holo-IsdX (grey). (B) Heme-binding pocket with stick representation of heme (carbon, blue and Fe, orange sphere), apo-IsdX1 residues (carbon, pink) and holo-IsdX1 residues (carbon, grey), and sulfur (yellow), nitrogen (blue) and oxygen (red). Arg-54 in the holo structure had two conformations and the alternate conformation has cyan carbon atoms.</p

Crystallography statistics.

a<p>Values for the highest resolution shell are shown in parentheses.</p>b<p>R_merge = Σ|I_hkl−hkl>|/ΣI_hkl, where I is the observed intensity for reflection hkl, and <i> is the mean intensity.</i></p><i>c<p>R_work = Σ||F_o(hkl)|−|F_c(hkl)||/Σ|F_o(hkl)|; R_free is calculated in the same way with 5–10% of reflections excluded from refinement.</p></i

Functional role of the 3₁₀-helix and adjacent residues: hemoglobin association.

<p>Wild-type (A), S52A (B), S53A (C), R54A (D), or M55A (E) IsdX1 were infused at 100, 200, 300, 350, or 500 nM over holo or apo-hemoglobin (wild-type only, panel F) coupled to a CM5 chip and response units recorded over 800 seconds. The dissociation constants (in nanomolar) were as follows: wild-type = 15.0±0.07, S52A = 14.0±0.17, S53A = 139.0±2.5, R54A = 5, 500±1, 800, and M55A = 18.0±0.4. Due to the weak response of R54A, the <i>K_D</i> was calculated from response curves using concentrations approximately 100 times that injected for the wild-type protein. All other dissociation constants represent the mean and standard deviation of three independent measurements for the injection of 300 nM (final concentration) IsdX1 (χ²</p