Narrow Results

We review contributions made towards the elucidation of CO and O₂ binding geometries in respiratory proteins. Nuclear magnetic resonance, infrared spectroscopy, Mössbauer spectroscopy, X-ray crystallography and quantum chemistry have all been used to investigate the Fe–ligand interactions. Early experimental results showed linear correlations between ¹⁷O chemical shifts and the infrared stretching frequency (ν_CO) of the CO ligand in carbonmonoxyheme proteins and between the ¹⁷O chemical shift and the ¹³CO shift. These correlations led to early theoretical investigations of the vibrational frequency of carbon monoxide and of the ¹³C and ¹⁷O NMR chemical shifts in the presence of uniform and non-uniform electric fields. Early success in modeling these spectroscopic observables then led to the use of computational methods, in conjunction with experiment, to evaluate ligand-binding geometries in heme proteins. Density functional theory results are described which predict ⁵⁷Fe chemical shifts and Mössbauer electric field gradient tensors, ¹⁷O NMR isotropic chemical shifts, chemical shift tensors and nuclear quadrupole coupling constants (e²qQ/h) as well as ¹³C isotropic chemical shifts and chemical shift tensors in organometallic clusters, heme model metalloporphyrins and in metalloproteins. A principal result is that CO in most heme proteins has an essentially linear and untilted geometry (τ = 4 °, β = 7 °) which is in extremely good agreement with a recently published X-ray synchrotron structure. CO/O₂ discrimination is thus attributable to polar interactions with the distal histidine residue, rather than major Fe–C–O geometric distortions.

This communication is devoted to nature determination and quantification by PIXE of metals contained in proteins after their separation by PolyAcrylamide Gel Electrophoresis (PAGE). After the electrophoresis, the gel is dried and each track is scanned with a 2.5 MeV proton beam which triggers metal X-ray fluorescence and then, allows to determine the type of metals contained in an electrophoretic band. For quantitative determination of the amount of the metal contained inside the band, the characteristic X-ray peak area is compared with those obtained with polyacrylamide gels doped with the same metal. The normalization has been achieved by using RBS measurements on the gel itself.

The procedure presented seems to be a very useful multielementary method for the metal content analysis and for the determination of the metal amounts inside proteins after their separation by electrophoresis. Furthermore it allows to check if metals remain bound to proteins.