Optical Detection of Disordered Water within a Protein Cavity

Abstract

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by x-ray crystallography. Here we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well defined, non-coordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring non-specific hydration of the active sites of heme proteins.

Blocking the Gate to Ligand Entry in Human Hemoglobin

“…Tian et al (65,66) estimated that the rate of opening and closing of the His(E7) gate in Mb is on the order of 1-10 s Ϫ1 , which competes with internal geminate rebinding of CO and water entry but not with bimolecular binding from solvent (69,70). Thus, CO rebinding to WT MbCO after laser photolysis is a simple one-step process on microsecond to millisecond time scales.…”

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

“…17,20,29 The Hb-chain samples were prepared similarly except that the concentrations ranged from 10 to 100 μ M and cuvette path lengths ranged from 1 to 10 cm. The protein concentrations were varied over these ranges to ensure that the signals measured did not include contributions from chain homodimers and homotetramers.…”

“…17,20,29 Photolysis difference spectra were recorded over the visible band spectral region (500–650 nm) at 41 time delays spaced logarithmically from 20 ns to 20 ms after the laser pulse. Each time-delayed spectrum represented the average of ~1000 photolysis measurements.…”

“…19 Modulation of the water occupancy was found to be a critical factor by which distal pocket residue mutations affected ligand binding rate constants in the variants studied. The rate at which water enters the Mb pocket is limited by the time scale of ligand escape, ~0.5 μ s. 17,19,21 As expected from the steric constraint imposed by the size of the distal cavity, water entry does not precede ligand escape in wild-type Mb or any variant studied to date.…”

“…12,13 However, positional disorder may obscure the electron densities of internal waters in X-ray diffraction studies, and orientational disorder and short residence times may similarly limit NMR detection. 1,14–17 Thus, the extent to which water may be buried in the internal cavities of proteins has remained an open question, although computational modeling may be helpful in this regard. 16,18 …”