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
Mr. Cheesman et al., 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, European journal of biochemistry, 219(1-2), 1994, pp. 595-602
Room-temperature (295 K) magnetic-circular-dichroism spectra at 280-25
00 nm have been recorded for Escherichia coli cytochrome bo in its fas
t form (which has a g = 3.7 EPR signal and reacts rapidly with cyanide
) and for its formate, fluoride, cyanide and hydrogen-peroxide derivat
ives. 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-tran
sfer band) at 1610 nm. High-spin ferric heme o gives rise to a negativ
e magnetic-circular-dichroism feature at 635, 642 and 625 nm (correspo
nding to a shoulder observed in the electronic absorption spectral 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 en
ergies of the analogous bands in the spectrum of fast cytochrome bo ar
e typical for high-spin ferric hemes with histidine and water axial li
gands. Addition of cyanide ion to fast cytochrome bo causes a red shif
t in the position of the Soret absorption peak, from 406.5 nm to 413 n
m, and results in the loss of the 635-nm feature from the magnetic-cir
cular-dichroism spectrum and of the corresponding shoulder in the elec
tronic absorption spectrum. In the magnetic-circular-dichroism spectru
m, 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-in
frared charge-transfer band suggests that the cyanide ion is bridged t
o the Cu-B of the binuclear site. Treatment of fast cytochrome bo with
hydrogen peroxide also causes a red shift in the position of the Sore
t 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 th
e magnetic-circular-dichroism Soret band and no new near-infrared char
ge-transfer bands are detected, ruling out a similar high-spin to low-
spin transition at heme o. Subtraction of the contribution of low-spin
ferric heme b from the magnetic-circular-dichroism spectrum reveals a
spectrum characteristic of ferryl [Fe(IV)I heme o.