title: Geometric structure determination of N694C lipoxygenase: a comparative near-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure study.
abstract: The mononuclear nonheme iron active site of N694C soybean lipoxygenase (sLO1) has been investigated in the resting ferrous form using a combination of Fe-K-pre-edge, near-edge (using the minuit X-ray absorption near-edge full multiple-scattering approach), and extended X-ray absorption fine structure (EXAFS) methods. The results indicate that the active site is six-coordinate (6C) with a large perturbation in the first-shell bond distances in comparison to the more ordered octahedral site in wild-type sLO1. Upon mutation of the asparagine to cysteine, the short Fe-O interaction with asparagine is replaced by a weak Fe-(H(2)O), which leads to a distorted 6C site with an effective 5C ligand field. In addition, it is shown that near-edge multiple scattering analysis can give important three-dimensional structural information, which usually cannot be accessed using EXAFS analysis. It is further shown that, relative to EXAFS, near-edge analysis is more sensitive to partial coordination numbers and can be potentially used as a tool for structure determination in a mixture of chemical species.
authors.affiliation: Stanford Synchrotron Radiation Laboratory, SLAC, Stanford University, Stanford, California 94309, USA.
authors.forename: Ritimukta
authors.initials: R
authors.lastname: Sarangi
authors.forename: Rosalie K
authors.initials: RK
authors.lastname: Hocking
authors.forename: Michael L
authors.initials: ML
authors.lastname: Neidig
authors.forename: Maurizio
authors.initials: M
authors.lastname: Benfatto
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
mesh_terms: D015502:Absorptiometry, Photon / Q000379:methods*; D019943:Amino Acid Substitution; D001216:Asparagine; D020134:Catalytic Domain; D003545:Cysteine; D006860:Hydrogen Bonding; D007287:Inorganic Chemicals / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013025:Glycine max / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D007287:Inorganic Chemicals; D001216:Asparagine; D008084:Lipoxygenase; D003545:Cysteine