Effect of ligands on cytochromed fromAzotobacter vinelandii

Abstract

Spectra of oxidized and reduced cytochromed in particles ofA. vinelandii were studied in the presence of the ligands CO, azide, and NH₂OH under oxidizing, reducing, and turnover conditions. Under oxidizing conditions, spectral changes were observed on oxidized cytochromed (absorption maximum at 648 nm) in the presence of CO and NH₂OH showing a shift of the maximum to shorter wavelengths (639 and 645 nm, respectively) and a broadening of the half-band width. Under reducing conditions, spectral changes were observed on reduced cytochromed (absorption maximum at 631 nm) in the presence of CO (absorption maximum at 636 nm), NO, NO⁻ ₂, and NH₂OH (absorption maximum at 642 nm in the presence of dithionite). The spectral changes of cytochromed in the presence of NH₂OH or with dithionite and NO⁻ ₂ were ascribed to the formation of the NO-cytochromed compound. Under turnover conditions CO, NH₂OH, and azide cause a spectral shift of the absorption maximum of cytochromed from 648 nm to 636, 645, and 655 nm, respectively. With NH₂OH and azide a broadening of the half-band width of 7 and 6 nm, respectively, was also observed. The spectral changes caused by CO and NH₂OH were interpreted as a binding of the ligands to cytochromed changing its conformation from the oxidized state absorbing at 648 nm into a more stable liganded form. Since azide does not affect the spectral bands of oxidized and reduced cytochromed, the spectral change during turnover in the presence of azide were ascribed to a preferential binding of azide to enzymically active conformation of cytochromed (cytochromed _x).