The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 ms. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the Ca atoms of G60 at switch 2 and G10 at the P-loop and the N-Ca-C-O dihedral (j) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/j plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ i

Biochemical and Mechanistic Studies of Nitronate Monooxygenase and Roles of Histidine Residues in Select Flavoprotein Oxidases

Nitronate monooxygenase (NMO) catalyzes the flavin-dependent oxidation of propionate 3-nitronate (P3N) via the formation of an anionic flavosemiquinone. The oxidation of substrate includes the formation of a peroxy-nitro acid intermediate. P3N is activated to its radical form via a single electron transfer onto the FMN cofactor forming the anionic flavosemiquinone. Reoxidation of FMN cofactor from the anionic semiquinone has been proposed to go through two routes dependent upon which radical species oxygen reacts with first, radical P3N or the semiquinone. The recent crystallographic determination of NMO from Cyberlindnera saturnus and steady-state kinetics revealed an allosteric activation effect on the enzyme by PEG 3350 with respect to P3N. Choline oxidase (CHO) catalyzes the two-step oxidation of choline to glycine betaine via an enzyme-bound FAD cofactor. In the first redox reaction, choline is activated to it alkoxide form by means of an enzyme-derived catalytic base, H466. This histidine residue has been shown to not only act as a general base but an electrostatic catalyst stabilizing the negative charge accumulated on the reduced flavin species as shown by replacing the residue with alanine, aspartate, and glutamine using site-directed mutagenesis. CHO was also observed to catalyze excited state reactions as facilitated by H466. Evidence for the ESR comes from the observation of a pL-dependence on the fluorescence emission of CHO in H2O and D2O. Using fluorescence spectroscopy and pH effects, a hydroxy-C4a flavin intermediate was detected in the wild-type and S101A variant with and without oxygen indicating the adduct formation was with an active site hydroxide ion. The mechanism of formation has been elucidated