title: Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.
abstract: The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.
authors.affiliation: †Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, United States.
authors.forename: Natalia
authors.initials: N
authors.lastname: Stein
authors.affiliation: †Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Love
authors.forename: Evan T
authors.initials: ET
authors.lastname: Judd
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Elliott
authors.affiliation: §Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.affiliation: †Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, United States.
authors.forename: A Andrew
authors.initials: AA
authors.lastname: Pacheco
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000187:drug effects; D045263:Cytochromes a1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003575:Cytochromes c1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology*; D012977:Sodium Nitrite / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013053:Spectrophotometry; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008024:Ligands; D050505:Mutant Proteins; D011994:Recombinant Proteins; D006418:Heme; D045263:Cytochromes a1; D003575:Cytochromes c1; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome); D012977:Sodium Nitrite; D011190:Potassium Cyanide