Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy

Abstract

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.

FIGURE 1

Fe K-edge XANES spectra of met-myoglobin at pH 7 at T = 70 K, before (Mb(III)H₂O, dashed curve), and after prolonged x-ray irradiation at high intensity (Mb(II)H₂O, solid curve), The last spectrum (solid curve) probes the Fe-heme structure in the trapped nonequilibrium state. The inset contains a blowup of the pre-edge features, indicating the formation of a low-spin Fe-heme adduct.

FIGURE 2

Upper curves are ɛ//a^* (dotted curve) and ɛ//c (solid curve) polarized spectra of Mb(II)H₂O in a single crystal at T = 70 K. Lower curves are the comparison between the spectra of Mb(II)H2O in solution (solid curve) and in the single crystal (dotted curve). This last spectrum is the 0.4/0.6 linear combination of the ɛ//a^* and ɛ//c spectra, respectively.

FIGURE 3

Temperature-dependence of the XANES spectra of photo-reduced myoglobin Mb(II)H₂O. (Left frame) ɛ//a^* spectra, polarized along the Fe-water bond. The thermally activated water dissociation is probed by the spectral changes of the ɛ//a^* spectrum between 200 K and 240 K. (Right frame) ɛ//c spectra, polarized on the heme plane. Here, the relaxation of the Fe-heme system, with the displacement of the iron from the heme plane, is probed between 160 K and 240 K. Between 120 K and 160 K, an evidence for a relaxation of the intermediate Mb(II)H₂O is observed in both the polarizations.

FIGURE 4

Comparison between the first state Mb(II)H₂O at T = 70 K and the final state Mb(II)⋯H2O at T = 240 K of the temperature-dependent dissociation process. (Upper frame) Polarization ɛ//a^* along the Fe-water bond. (Middle frame) Polarization ɛ//c on the Fe-heme plane. (Lower frame) Comparison between the spectra of the deoxy-Mb(II) in solution obtained by reduction of met-myoglobin with sodium dithionite (solid curvee) and the Mb(II)—H₂O final state of our experiment at T = 240 K in single crystal (dotted curve). This last spectrum is the 0.4/0.6 linear combination of the ɛ//a^* and ɛ//c spectra, respectively.

FIGURE 5

Best fit of the XANES spectra of Mb(III)H₂O and Mb(II)H₂O in solution at T = 70 K in terms of the selected structural parameters reported in Tables 1 and 2 (solid lines). The experimental data are plotted in circles.

FIGURE 6

Best fit of the polarized XANES spectra of Mb(II)H₂O at T = 70 K and Mb(II)⋯H2O at T = 240 K in terms of the selected structural parameters reported in Tables 2 and 3 (solid lines). The experimental data are plotted in circles.

FIGURE 7

Sketch of the structure of the three main states. The structural data extracted by XANES are reported together with their statistical errors.