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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. Biochemistry 2015 Jun 23;54(24):3749-58

Date

06/05/2015

Pubmed ID

26042961

Pubmed Central ID

PMC4743497

DOI

10.1021/acs.biochem.5b00330

Scopus ID

2-s2.0-84935012019 (requires institutional sign-in at Scopus site)   10 Citations

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.

Author List

Stein N, Love D, Judd ET, Elliott SJ, Bennett B, Pacheco AA

Authors

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University
Arsenio Andrew Pacheco in the Chemistry and Biochemistry department at University of Wisconsin - Milwaukee




MESH terms used to index this publication - Major topics in bold

Amino Acid Substitution
Bacterial Proteins
Catalytic Domain
Cytochromes a1
Cytochromes c1
Electron Spin Resonance Spectroscopy
Enzyme Inhibitors
Heme
Ligands
Models, Molecular
Molecular Conformation
Mutagenesis, Site-Directed
Mutant Proteins
Nitrate Reductases
Oxidation-Reduction
Potassium Cyanide
Protein Conformation
Recombinant Proteins
Shewanella
Sodium Nitrite
Spectrophotometry
Titrimetry