Magnetic-circular-dichroism studies of Escherichia coli cytochrome bo. Identification of high-spin ferric, low-spin ferric and ferryl [Fe(IV)] forms of heme o.
Room-temperature (295 K) magnetic-circular-dichroism spectra at 280-2500 nm have been recorded for Escherichia coli cytochrome bo in its fast form (which has a g = 3.7 EPR signal and reacts rapidly with cyanide) and for its formate, fluoride, cyanide and hydrogen-peroxide derivatives. The spectra of all forms are dominated by signals from low-spin ferric heme b. These include a porphyrin-to-ferric ion charge-transfer transition in the near-infrared region (the near-infrared charge-transfer band) at 1610 nm. High-spin ferric heme o gives rise to a negative magnetic-circular-dichroism feature at 635, 642 and 625 nm (corresponding to a shoulder observed in the electronic absorption spectra) and a derivative charge-transfer feature at 1100, 1180 and 940 nm for the fast, formate and fluoride forms, respectively. The energies of these bands confirm that fluoride and formate are ligands to heme o. The energies of the analogous bands in the spectrum of fast cytochrome bo are typical for high-spin ferric hemes with histidine and water axial ligands. Addition of cyanide ion to fast cytochrome bo causes a red shift in the position of the Soret absorption peak, from 406.5 nm to 413 nm, and results in the loss of the 635-nm feature from the magnetic-circular-dichroism spectrum and of the corresponding shoulder in the electronic absorption spectrum. In the magnetic-circular-dichroism spectrum, the intensities of the Soret and alpha, beta bands are significantly increased. New near-infrared charge-transfer intensity is observed at 1000-2300 nm with a peak near 2050 nm. These changes are interpreted as resulting from a high-spin to low-spin transition at ferric heme o brought about by the binding of cyanide ion. The energy of the near-infrared charge-transfer band suggests that the cyanide ion is bridged to the CuB of the binuclear site. Treatment of fast cytochrome bo with hydrogen peroxide also causes a red shift in the position of the Soret absorbance, to 412 nm, and a loss of the 625-nm absorption shoulder. Changes in the magnetic-circular-dichroism spectrum at 450-600 nm are observed, but there is no significant increase in the intensity of the magnetic-circular-dichroism Soret band and no new near-infrared charge-transfer bands are detected, ruling out a similar high-spin to low-spin transition at heme o.(ABSTRACT TRUNCATED AT 400 WORDS)
Cheesman
M R
MR
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, England.
Watmough
N J
NJ
Gennis
R B
RB
Greenwood
C
C
Thomson
A J
AJ
D002942:Circular Dichroism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006418:Heme / Q000032:analysis*; D006861:Hydrogen Peroxide; D007501:Iron / Q000032:analysis*; D008280:Magnetics; D011487:Protein Conformation; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C069808:heme O; D006418:Heme; C017168:cytochrome bo, E coli; D006861:Hydrogen Peroxide; D007501:Iron