Cytochrome c-552 from Gram-Negative Alkaliphilic Pseudomonas alcaliphila AL15-21~T Alters the Redox Properties at High pH

摘要

A soluble class I cytochrome c of an alkaliphile was purified and characterized,and its primary structure was determined.This is the first example of a soluble class I cytochrome c in alkali-philes.Cells the alkaliphilic gram-negative bacterium Pseudomonas alcaliphila AL15-21~T grown at pH 10 had a soluble cytochrome c content that was more than twofold that of strain AL15-21~T cells grown at pH 7 under air-limited conditions.Cytochrome c-552,a soluble cytochrome c with a low molecular weight,was purified from strain AL15-21~T cells grown at pH 10 under air-limited conditions.Cytochrome c-552 had a molecular mass of 7.5 kDa and exhibited an almost fully reduced state in the resting form,which exhibited absorption maxima at wavelengths of 552,523 and 417 nm.In the oxidized state,it exhibited an absorption maximum at 412 nm when it was oxidized by ferricyanide,its isoelectric point(pI)was 4.3 and it contained one heme c as a prosthetic group.Cytochrome c-552 was autoreduced at pH 10,and the autoreduction was reproducible.On the other hand,the autoreduction of cytochrome c-552 was not observed at pH 7.0.When pH was increased from 7.0 to 8.3,its midpoint redox potentials(E_m values)increased from +228 mV to +276 mV as determined by redox titrations,and from +217 mV to +275 mV as determined by cyclic voltammetric measurements.The amino acid sequence deduced by cytochrome c-552 gene analysis revealed that the sequence consists of 96 residues,including 19 residues as an amino-ter-minal signal peptide.A phylogenetic tree based on amino acid sequence indicated that the protein belongs to group 4,cytochrome c5 in class I cytochrome c.