Isoelectrophoretic characterization of Pseudomonas cytochrome oxidase/nitrite reductase and its heme d1-containing domain.
The cytochrome oxidase/nitrite reductase of Pseudomonas aeruginosa has been purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When this "homogeneous" protein is subjected to electrophoretic titration curve analysis in ampholines or to isoelectric focusing in immobilized pH gradient gels it is resolved into several bands, each of which possesses the olive-green color of the holoenzyme. Although the patterns of resolution replicate for a given enzyme preparation differences occur among different preparations. Furthermore, storage for several months at -20 degrees C leads to an increase in the number of isoelectrophoretic forms. All preparations, however, have two primary bands, one with a pI of 6.97 and the other of 7.02. Both these bands possess significant cytochrome oxidase activity after elution from the gels. When each of the primary bands is eluted and again subjected to isoelectric focusing under the same conditions as before, each band interconverts into two bands with pIs of 6.97 and 7.02. The addition of the ligand cyanide to the holoenzyme produces a shift in the pI of the two bands to pIs 7.04 and 7.12 while the addition of nitrite shifts some of the band at pI 6.97 into that at pI 7.02. The heme d1-containing dipeptide of the enzyme, produced by treatment with subtilisin, also exhibits considerable heterogeneity upon electrophoretic titration curve analysis and by isoelectric focusing in immobiline gels. Possible explanations for the observed isoelectrophoretic behavior in terms of protein conformation and heme chemistry are discussed.
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
H H
HH
Hull
D C
DC
Wharton
D000595:Amino Acid Sequence; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification*; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives* / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009572:Nitrite Reductases / Q000737:chemistry / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013381:Subtilisins / Q000494:pharmacology
D006418:Heme; C029400:heme d1; D009572:Nitrite Reductases; D003576:Electron Transport Complex IV; D013381:Subtilisins