title: Spectroscopic and kinetic characterization of the recombinant wild-type and C242S mutant of the cytochrome b reductase fragment of nitrate reductase.
abstract: Spectroscopic and kinetic studies comparing the behavior of the recombinant cytochrome b reductase fragment of corn leaf nitrate reductase and a mutant in which cysteine 242 is replaced with a serine residue (C242S) have been carried out. The visible and circular dichroism spectra of the wild-type and mutant protein are virtually identical and compare well with those reported for nitrate reductases from other sources. The reduced wild-type protein forms a charge-transfer complex with NAD+ that has an absorption envelope that extends into the near infrared, with a maximum around 800 nm. The C242S mutant forms a similar charge-transfer complex with NAD+ but to a lesser extent than the wild-type. The reduction potential of the flavin for the wild-type protein is -287 mV, and that for the mutant is -279 mV. The rate of reduction by NADH of the C242S mutant is 7-fold slower than that for the wild-type protein, and the Kd is larger by a factor of 2. These results indicate that the cysteine 242 residue plays a role principally in facilitating electron transfer from NADH to the flavin rather than in binding of NADH to the enzyme.
authors.affiliation: Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ratnam
authors.forename: N
authors.initials: N
authors.lastname: Shiraishi
authors.forename: W H
authors.initials: WH
authors.lastname: Campbell
authors.forename: R
authors.initials: R
authors.lastname: Hille
mesh_terms: D002942:Circular Dichroism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D003313:Zea mays / Q000201:enzymology*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D009243:NAD; D009565:Nitrate Reductases; D050901:Nitrate Reductase