Internal water and microsecond dynamics in myoglobin

Abstract

Myoglobin (Mb) binds diatomic ligands, like O2, CO, and NO, in a cavity that is only transiently accessible. Crystallography and molecular simulations show that the ligands can migrate through an extensive network of transiently connected cavities but disagree on the locations and occupancy of internal hydration sites. Here, we use water (2)H and (17)O magnetic relaxation dispersion (MRD) to characterize the internal water molecules in Mb under physiological conditions. We find that equine carbonmonoxy Mb contains 4.5 ± 1.0 ordered internal water molecules with a mean survival time of 5.6 ± 0.5 μs at 25 °C. The likely locations of these water molecules are the four polar hydration sites, including one of the xenon-binding cavities, that are fully occupied in all high-resolution crystal structures of equine Mb. The finding that water escapes from these sites, located 17-31 Å apart in the protein, on the same μs time scale suggests a global exchange mechanism. We propose that this mechanism involves transient penetration of the protein by H-bonded water chains. Such a mechanism could play a functional role by eliminating trapped ligands. In addition, the MRD results indicate that 2 or 3 of the 11 histidine residues of equine Mb undergo intramolecular hydrogen exchange on a μs time scale.

Figure 1

Crystal structure1DWR of equine MbCO in (a) ribbon and (b) cross-sectional surface (1.4 Å probe) representations, rendered with PyMol (Schrödinger, LLC). Color code: heme (orange), CO (cyan), internal water molecules (red), and xenon binding sites (green).

Figure 2

(a) ²H MRD profile, R₁(ω₀), from immobilized MbCO at 25 °C, pD 7.0, and N_GA = 29.9, scaled to N_W = 3 × 10⁴. The error bars do not exceed the symbol size. The solid curve is the fit that resulted in the parameter values in Table 1. The dashed curves are dispersion components 1 (magenta), 2 (blue), and 3 (red). The bulk water (black dotted) and external hydration shell (black dashed) contributions are also shown. (b) Zero-frequency excess relaxation rate, R₁^ex(0), obtained from fits to 22 MRD profiles, versus pD. The red curve is a fit based on the histidine pK_a values in Mb, and the dashed line is the deduced pD-independent water contribution. The difference between the two curves hence represents the His LD contribution. Solid black symbols represent different buffer conditions as indicated. Colored symbols refer to different MbCO concentrations: 2.65 mM (green solid circle), 1.35 mM (green open circle), or 1.02 mM (blue solid circle); and N_GA: 110 (blue solid circle) or 62.1 (blue open circle). In all other cases, C_Mb = 1.60–1.67 mM and N_GA = 29.7–31.1.

Figure 3

(a) ²H MRD profile, R₁(ω₀), from free-tumbling MbCO at 25 °C and pD 7.0, scaled to N_W = 3 × 10⁴. The error bars are up to twice the symbol size. The solid curve is a single-component fit to the solid data points (ω₀/2π > 0.9 MHz), with the parameters in Table 1. The bulk water (dotted line) and external hydration shell (dashed line) contributions are shown. (b) Amplitude NS_iso² (red squares) and correlation time τ_C (blue circles), obtained from single-component fits to five MRD profiles, versus pD. The dotted lines are visual guides.

Figure 4

(a) ¹⁷O MRD profile, R₁(ω₀), from MbCO gel at 25 °C, pD 7.0, and N_GA = 29.9, scaled to N_W = 3 × 10⁴. The error bars are up to twice the symbol size. The solid curve is the fit that resulted in the parameter values in Table 1. The dashed curves are dispersion components 1 (magenta), 2 (blue), and 3 (red). The bulk water (dotted line) and external hydration shell (dashed line) contributions are shown in both panels. (b) ¹⁷O MRD profile, R₁(ω₀), from MbCO solution at 25 °C and pD 7.0, scaled to N_W = 3 × 10⁴. The error bars are comparable to the symbol size. The solid curve is a single-component fit, with the parameters in Table 1.

Figure 5

²H MRD profile, R₁(ω₀), from immobilized MbCO (25 °C, pD 7.16, scaled to N_W = 3 × 10⁴) in the presence (blue symbols) or absence (red symbols) of 8 bar of xenon. The error bars do not exceed the symbol size. The parameter values obtained from the fits (solid red and dashed blue curves) are collected in Table S5 (Supporting Information).