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: 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

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

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

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