Browsing by Author "Dooley, David M."

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Kβ X-ray Emission Spectroscopy as a Probe of Cu(I) Sites: Application to the Cu(I) Site in Preprocessed Galactose Oxidase
(American Chemical Society, 2020-11) Lim, Hyeongtaek; Baker, Michael L.; Cowley, Ryan E.; Kim, Sunghee; Bhadra, Mayukh; Siegler, Maxime A.; Kroll, Thomas; Sokaras, Dimosthenis; Weng, Tsu-Chien; Biswas, Dalia R.; Dooley, David M.; Karlin, Kenneth D.; Hedman, Britt; Hodgson, Keith O.; Solomon, Edward I.
Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kβ X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kβ XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double-ionization (DI) states are identified using varying incident X-ray photon energies, and a reasonable method to correct the data to remove DI contributions is presented. Kβ XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kβ2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kβ2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d–p mixing. A Kβ XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kβ2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2–y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kβ XES in probing the Cu(I) FMO associated with small-molecule activation in metalloproteins.
Spectroscopic Identification of Heme Axial Ligands in HtsA That Are Involved in Heme Acquisition by Streptococcus pyogenes
(2010-04) Ran, Yanchao; Liu, Mengyao; Zhu, Hui; Nygaard, Tyler K.; Brown, Doreen E.; Fabian, Marian; Dooley, David M.; Lei, Benfang
The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV−vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV−vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV−vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a Kd of 40.9 μM but not with H229A HtsA, and cyanide binds to M79A and H229A with Kd of 0.5 and 19.1 μM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.