A Double Peak c-Type Cytochrome, Cytochrome c-552, 558 of a Denitrifying Bacterium, Pseudomonas stutzeri

Abstract

From acetone-dried powder of aerobically grown cells of a denitrifying bacterium, van Niel strain of Pseudomonas stutzeri, a new c-type cytochrome, which exhibits absorp-tion spectra and chemical properties different from those of other two c-type cytochromes contained in the same organism, could be obtained in highly purified state and was referred to as cytochrome c-552, 558.
In 0.05M phosphate buffer, pH 7.0, absorption maxima of reduced cytochrome c-552, 558 are at 421, 526, and 558mμ with a shoulder at 552mμ. The rnillimolar extinction coefficient (based on the heme content) at 558 mp is very low (ε_mM=18.8). In the presence of alkali, urea and alcohols, the reduced spectrum is converted to that of typical bacterial c-type cytochrome : absorption maxima appear at 418, 524, and 552mμ and the absorbancy at each maximum is greatly intensified. The spectrum thus changed is reversed to its original state on removing these reagents.
Reduced cytochrome c-552, 558 is oxidized in air and reacts with CO even at neutral pH. Oxidized hemoprotein can be reduced by dithionite, but not by borohydride, ascorbate or cysteine.
The molecular weight of cytochrome c-552, 558 estimated from centrifugal data or a gel-filtration method is about 70, 000 and its minimum molecular weight calculated from the heme content is about 37, 000, indicating that it contains two hemes per molecule.
Cytochrome c-552, 558 was found to be also contained in the soluble fraction on disruption of washed fresh cells by sonic oscillation followed by centrifugation at 105, 000×g for 1 hr.