title: Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin.
abstract: Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Katlyn K
authors.initials: KK
authors.lastname: Meier
authors.forename: Shiliang
authors.initials: S
authors.lastname: Tian
authors.forename: Jun-Long
authors.initials: JL
authors.lastname: Zhang
authors.forename: Hongchao
authors.initials: H
authors.lastname: Guo
authors.forename: Charles E
authors.initials: CE
authors.lastname: Schulz
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Nilges
authors.forename: Eckard
authors.initials: E
authors.lastname: Münck
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D018360:Crystallography, X-Ray; D005296:Ferrous Compounds / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005296:Ferrous Compounds; D019159:Nonheme Iron Proteins; D013481:Superoxides; D001400:Azurin; D003300:Copper