Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6

Registration of ‘Darrell’ Wheat

‘Darrell’ (Reg. No. CV-1024, PI 644224) hard red winter wheat (Triticum aestivum L.) was developed by the South Dakota State University–Agricultural Experiment Station and released in 2006 to seed producers by the South Dakota State University–Agricultural Experiment Station and the University of Nebraska Agricultural Experiment Station. Darrell was selected from the cross 2076-W12-11/‘Karl 92’ (PI 564245)//NE89526. NE89526 is an experimental line from the University of Nebraska with the pedigree ‘Lancota’ (CI 17389) selection/’Siouxland’ (PI 483469)//TX792729. 2076-W12-11 is an unreleased experimental line that originated from a population with an unidentified pedigree. Darrell was selected as an F3:4 line in 1998 and was assigned experimental line number SD98102. Darrell was released on the basis of its good disease resistance and excellent yield potential in the northern Great Plains

Structural and electronic properties of the heme cofactors in a multi- heme synthetic cytochrome

Resonance Raman, absorption, and electron paramagnetic resonance spectra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome b maquette described by Robertson et al. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is composed of 62 amino acid residues arranged in a di-α-helical unit which dimerizes in solution to form a four-helix bundle. Each di-α-helical unit contains histidine residues at the 10,10\u27 positions which serve as ligands to the hemes. When protoheme IX is incorporated, both hemes in the dimer are bis- ligated and low spin. The two hemes are inequivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studies were conducted on peptides reconstituted with protoheme IX (PHa) and several related variants. These hemes include 2-vinyldeuteroheme (2-VDH), 4-vinyldeuteroheme (4-VDH), protoheme III (PHs), and 1-methyl-2-oxomesoheme XIII (2-OMH). Collectively, the spectroscopic studies reveal the following: (1) 2-VDH, 4-VDH, and 2-OMH bind to the protein and form bis-ligated low-spin complexes similar to PHa. The structures of the two hemes in the dimers are identical as are the immediate protein environments around the bound cofactors. These results indicate that the redox inequivalence of the two hemes is due to heme-heme electronic interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arranged in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the vinyl group(s) are in the hydrophobic interface between the two units which comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs protein differs from the other synthetic proteins in that it contains one pentacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination site on the high- spin heme is inaccessible to exogenous imidazole but readily binds cyanide, suggesting that the α-helix containing the unbound histidine is nearby and partially shields the coordination site. The high-spin heme converts to low- spin at low-temperature, presumably via binding of the histidine residue on this nearby α-helix. It is suggested that the different behavior observed for the PHs protein is due to the fact that this heme is symmetric with respect to rotation about the α,γ-axis of the macrocycle which bisects the meso-carbons between the vinyl groups and propionic acid residues. This symmetry precludes rotational isomerism about the α,γ-axis to establish an unhindered fit. In contrast, all the other hemes examined contain at least one substituent smaller than a vinyl group which together with the fact that two different α,γ-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid the like-substituent-like-substituent heme-heme interactions of PHs. The propensity to avoid such interactions could explain the inequivalent binding properties of the two hemes in the dimer. For the PHs protein wherein these interactions cannot be mitigated by rotation of the heme, other rearrangements of the protein must occur. These rearrangements could force the second-bound heme to assume a high-spin configuration

Registration of ‘Darrell’ Wheat

‘Darrell’ (Reg. No. CV-1024, PI 644224) hard red winter wheat (Triticum aestivum L.) was developed by the South Dakota State University–Agricultural Experiment Station and released in 2006 to seed producers by the South Dakota State University–Agricultural Experiment Station and the University of Nebraska Agricultural Experiment Station. Darrell was selected from the cross 2076-W12-11/‘Karl 92’ (PI 564245)//NE89526. NE89526 is an experimental line from the University of Nebraska with the pedigree ‘Lancota’ (CI 17389) selection/’Siouxland’ (PI 483469)//TX792729. 2076-W12-11 is an unreleased experimental line that originated from a population with an unidentified pedigree. Darrell was selected as an F3:4 line in 1998 and was assigned experimental line number SD98102. Darrell was released on the basis of its good disease resistance and excellent yield potential in the northern Great Plains