Dataset Information

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase.

ABSTRACT: Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOS oxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOS oxy-NO, demonstrating the strong trans effect of the heme-bound NO.

SUBMITTER: Horn M

PROVIDER: S-EPMC4304226 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase.

Horn Michael M Nienhaus Karin K Nienhaus Gerd Ulrich GU

F1000Research 20141128

Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local ...[more]

PMID: 25653844

Similar Datasets

Project description:Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy.

Dataset Information

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase.

Publications

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets