Infrared and EPR Studies on Cyanide Binding to the Heme-Copper Binuclear Center of Cytochrome bo-type Ubiquinol Oxidase from Escherichia coli

Abstract

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding.

Direct electrochemistry of cytochrome bo3 oxidase at a series of gold nanoparticles-modified electrodes

“…[23] Upon addition of KCN in the buffer solution (figure 2), the catalytic wave is indeed shifted to more negative potentials and decreases continuously. This behavior confirms again that cytochrome bo 3 is responsible for the reduction of oxygen here.…”

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy

“…The interesting recognition phenomena are the binding of dioxygen in the binuclear reaction center and the transfer of electrons and protons to O 2 (Yamazaki et al, 1999). The binuclear reaction center has been extensively characterized also by FTIR using CO, NO, CN À and N 3 À as spectroscopic probes (Tsubaki et al, 1996(Tsubaki et al, , 1997Zhao et al, 1994). Depending on the redox state of the different redox centers within the bovine heart cytochrome c oxidase, the stretch frequency of the CO ligand is observed between 1959 and 1965 cm À1 (Yoshikawa et al, 1977;Yoshikawa and Caughey, 1982).…”

Glutamate‐286 mutants of cytochrome bo‐type ubiquinol oxidase from Escherichia coli: influence of mutations on the binuclear center structure revealed by FT‐IR and EPR spectroscopies1

“…2, bottom). For E286D, the 2170 cm −1 band, the Cu B ‐cyano complex released from the auto‐reduced enzyme [15], was observed much faster and more significantly than for the wild‐type enzyme (Fig. 2, bottom).…”

“…Heme and copper contents were analyzed as described previously [12, 15]. Heme content was calculated as a sum of hemes B and O using a molar extinction coefficient for heme B [12].…”