title: Electron self-exchange in hemoglobins revealed by deutero-hemin substitution.
abstract: Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction.
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Navjot Singh
authors.initials: NS
authors.lastname: Athwal
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Jagannathan
authors.initials: J
authors.lastname: Alagurajan
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Sturms
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: D Bruce
authors.initials: DB
authors.lastname: Fulton
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Andreotti
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, United States. Electronic address: msh@iastate.edu.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hargrove
mesh_terms: D000641:Ammonia / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D003903:Deuterium; D006427:Hemin / Q000031:analogs & derivatives* / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006736:Horses; D019811:Hydroxylamine / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009211:Myoglobin / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D012275:Oryza; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D046939:Synechocystis
chemical_list: D001426:Bacterial Proteins; D006454:Hemoglobins; D009211:Myoglobin; D009587:Nitrogen Isotopes; D010940:Plant Proteins; C011219:deuterohemin; D019811:Hydroxylamine; D006427:Hemin; D000641:Ammonia; D003903:Deuterium
keywords: Deuteroheme; Electron transfer; Hemoglobin; Hydroxylamine reduction; Plants