Interaction of dimeric horse cytochrome c with cyanide ion

Abstract

We have previously shown that methionine-heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10(4) M(-1). The Fe-CN and C-N stretching (ν (Fe-CN) and ν (CN)) resonance Raman bands of CN(-)-bound dimeric cytochrome c were observed at 443 and 2,126 cm(-1), respectively. The ν (Fe-CN) frequency of dimeric cytochrome c was relatively low compared with that of other CN(-)-bound heme proteins, and a relatively strong coupling between the Fe-C-N bending and porphyrin vibrations was observed in the 350-450-cm(-1) region. The low ν (Fe-CN) frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 °C increased from (8.9 ± 0.7) × 10(-6) to (3.8 ± 0.2) × 10(-5) s(-1), with a decrease of about 2 °C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…The Soret band of oxidized dimeric AA cyt c 555 red shifted to 413 nm and the 695 nm absorption band disappeared by an addition of 1 m M potassium cyanide at pH 7.0, although no change was observed in the spectra by the addition of potassium cyanide to the oxidized monomer under the same conditions. A similar spectral change has been observed in CN ‐ binding to oxidized dimeric horse cyt c . The absorption changes in the spectrum of oxidized dimeric AA cyt c 555 by an addition of CN − were also observed at 85°C; this was not the case in the oxidized monomer [Supporting Information Fig.…”

“…The structure around the heme‐ligating Met was not affected much and Met remained coordinated to the heme iron by the dimerization of HT cyt c 552 , where the hinge region was located away from the heme‐ligating Met . When the hinge region was located close to the heme‐ligating Met such as in horse cyt c , the structure around the heme‐ligating Met was perturbed and Met dissociated from the heme iron by the dimerization, causing increase compared with the monomer in the binding properties of external ligands, such as CN − and H 2 O 2 . For the case of AA cyt c 555 , the hinge region was located slightly more away from the heme‐ligating Met compared with that in horse cyt c owing to the extra 3 10 ‐α‐3 10 helix, which caused no dissociation of Met from the heme iron in the dimer crystal but increase in the ligand binding properties (Figs.…”

“…The Met ligand dissociated from the heme iron in the horse cyt c dimer, whereas it was coordinated to the heme iron in the HT cyt c 552 dimer. CN ‐ and peroxides have been shown to react with dimeric horse cyt c , owing to dissociation of Met80 from the heme iron . The region containing the extra 3 10 ‐α‐3 10 helix and C‐terminal helix was swapped in dimeric AA cyt c 555 , and Met61 in the extra 3 10 ‐α‐3 10 helix was coordinated to the heme iron in its crystal structure.…”

“…We have previously shown that horse cyt c forms oligomers by swapping its C‐terminal α‐helix intermolecularly, without changing its secondary structures significantly . Since Met80 dissociated from the heme iron in dimeric horse cyt c , CN − and H 2 O 2 bound to the heme iron easier in the dimer compared with the monomer . These results indicated that the properties of cyt c may be modified by domain swapping without changing its amino acid sequence, although the dimer may dissociate to monomers at high temperatures or acidic pH .…”

“…53 Since Met80 dissociated from the heme iron in dimeric horse cyt c, CN 2 and H 2 O 2 bound to the heme iron easier in the dimer compared with the monomer. 54,55 These results indicated that the properties of cyt c may be modified by domain swapping without changing its amino acid sequence, although the dimer may dissociate to monomers at high temperatures or acidic pH. 53 Cytochrome c 552 from thermophilic bacteria Hydrogenobacter thermophilus (HT cyt c 552 ) is also a thermostable protein belonging to the cyt c protein family.…”