Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin

Abstract

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

Figure 1

Visible band time-resolved photolysis difference spectra of isolated HbCO α chains at 20 °C. Spectra were measured at logarithmically distributed time delays: 18 time delays covering the range from 20 ns to 1 μs and 25 covering the range from 1.6 μs to 40 ms. Each time-delayed spectrum represented the average of ~1000 photolysis measurements.

Figure 2

Visible band time-resolved photolysis difference spectra of isolated HbCO β chains at 20 °C. Time delays are as described in the legend of Figure 1.

Figure 3

First two SVD components of CO photolysis TROA spectra in the visible bands of isolated Hb chains at 20 °C: (a and f) Mb (shown for comparison), (b and g) Hb α chains, (c and h) Hb β chains, (d and i) Hb αE7L chains, and (e and j) Hb βE7L chains. Panels a–e: (red) first spectral basis vector and (blue) second spectral vector, scaled by their singular values as indicated by the y-axis label. The second spectral vectors were multiplied by a factor of 20 for the sake of clarity. Panels f–i: (red) first temporal basis vector and (blue) second temporal basis vector, scaled by their singular values as indicated by the y-axis label. The second temporal basis vectors were multiplied by a factor of 25 for the sake of clarity.

Figure 4

b-Spectra (decay spectra) for the four-exponential fit to ligand photolysis of isolated HbCO α chains: (red) geminate CO rebinding, (blue) distal pocket water entry, (green) protein structural relaxation, and (dark red) bimolecular CO rebinding. Exponential lifetimes and relative spectral amplitudes are listed in Table 1. Spectra for water entry and protein structural relaxation are shown multiplied by a factor of 5 for the sake of visibility.

Figure 5

Visible band time-resolved photolysis difference spectra of the HbCO tetramer. Time delays are as described in the legend of Figure 1.

Figure 6

Visible band b-spectra (exponential decay spectra) for ligand photolysis of the human HbCO tetramer. The same spectra are shown in the inset normalized by their vector norms to better compare band shapes. The six observed kinetic phases (and their nominal assignments) in order of increasing lifetime (lifetimes and relative amplitudes listed in Table) are (1) (red) phase I (first CO geminate rebinding), (2) (light blue) phase II (second geminate rebinding), (3) (light green) phase III (initial R → T relaxation), (4) (dark green) phase IV (final R → T relaxation, competes with R₀ + CO bimolecular recombination), (5) (dark red) phase V (bimolecular rebinding to R-state tetramers), and (6) (black) phase VI (bimolecular rebinding to T-state tetramers). Nominal assignments were taken from previous multiwavelength TROA studies.^,– Spectra for phases III and IV are shown multiplied by a factor of 5 for the sake of visibility. Deviations of the band shapes (see the inset) for phases II–IV from those for pure CO rebinding (phase I) and/or R to T relaxation (green trace in Figure 4) indicate the presence of an additional process, distal pocket water entry, as was most evident for phase II.

Figure 7

Deconvolution of (black) b-spectra for kinetic phases I–VI (a–f, respectively) into components for (red) CO rebinding, (green) protein structural relaxation, (blue) distal pocket water entry, and (gray) baseline offset. The water entry and structural relaxation components are multiplied by the factors indicated in panels b and d–f for the sake of clarity. The sum of the deconvoluted spectral components (dotted line) is shown for comparison with the measured b-spectrum for each kinetic phase. The amplitudes of the deconvoluted components were used to calculate the corresponding component values listed in Table 2, as described in the text. Note that the signs of the water entry and protein relaxation signals are reversed in panels e and f from those shown in panels b–d, consistent with the loss of DP water and reversal of protein relaxation expected when bimolecular CO rebinding returns the protein to its prephotolysis state (see the text and Table 2).

Figure 8

Fractional concentrations of HbCO allosteric intermediates (see Scheme 1), calculated from the data in Tables 2 and 3, illustrate increased heme hydration as protein quaternary/tertiary conformations evolve in time (data for hydrated heme species are colored light and dark blue and data for anhydrous species gray): (gray, –·–) [Fe···CO]_Rr″, (gray, – – –) [Fe···]_Rr′, (light blue, – – –) [Fe···H₂O]_Rr′, (gray, – — –) [Fe···]_Rr, (light blue, – — –) [Fe···H₂O]_Rr, (gray, ···) [Fe···]_T′r, (dark blue, ···) [Fe···H₂O]_T′r, (gray, —) [Fe···]_Tt, and (dark blue, —) [Fe···H₂O]_Tt. Transition points in the time course of the total fractional population of deoxy hemes (black line) are labeled by the corresponding kinetic phases and their nominal assignments.

Figure 9

Time evolution of distal pocket water occupancies, n_w, in deoxy hemes of (light blue) R-state and (dark blue) T-state tetramers after CO photolysis, corresponding to the kinetic intermediate shown in Figure 8 (see the text). Dotted lines show asymptotic n_w values for the equilibrium R and T states. The black arrow indicates the increase in heme hydration during the R to T transition.

Scheme 1