Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.

Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.

Protein Dynamics by Two-Dimensional Infrared Spectroscopy.

Proteins function as ensembles of interconverting structures. The motions span from picosecond bond rotations to millisecond and longer subunit displacements. Characterization of functional dynamics on all spatial and temporal scales remains challenging experimentally. Two-dimensional infrared spectroscopy (2D IR) is maturing as a powerful approach for investigating proteins and their dynamics. We outline the advantages of IR spectroscopy, describe 2D IR and the information it provides, and introduce vibrational groups for protein analysis. We highlight example studies that illustrate the power and versatility of 2D IR for characterizing protein dynamics and conclude with a brief discussion of the outlook for biomolecular 2D IR.

Measuring the internal quantum yield of upconversion luminescence for ytterbium-sensitized upconversion phosphors using the ytterbium(iii) emission as an internal standard.

A method is presented for estimating the internal quantum yield (IQY) of NIR-to-NIR and NIR-to-visible upconversion (UC) luminescence for Yb3+-sensitized energy-transfer upconversion (ETU) phosphors. The method does not require an integrating sphere or a secondary standard, but rather uses the 1 µm emission of the Yb3+ sensitizer as an internal standard. The method requires the acquisition of the 1 µm emission decay curve of the UC phosphor using low pulse-energy density, an estimation of the radiative decay constant of the 1 µm emission, and emission spectra corrected for instrument response. This method is valid for UC emission spectra acquired via pulsed or continuous wave (cw) excitation. The method is demonstrated for cw excitation to obtain IQY for UC and downshifted luminescence for ß-phase NaYF4: 0.5%Tm, 25%Yb and NaYF4: 2%Er, 18%Yb nanocrystals (with and without a passivating NaYF4 shell) over a range of excitation irradiance. The corresponding results are consistent with those obtained using integrating spheres and numerical simulations, respectively. For pulsed excitation, an additional alternative method is described which requires acquisition of the 1 µm emission decay curve at each excitation pulse-energy density for which the IQY is to be determined. The proposed methods should be particularly useful for samples having very low absorbance at the excitation wavelength, for which direct determination methods are impractical.