Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component.
The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors. The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase. The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps. The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron. Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes. The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis. FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5. Nitrate and nitrite salts caused a drastic reduction in activity. Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen.
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
M J
MJ
Axley
D A
DA
Grahame
T C
TC
Stadtman
D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010100:Oxygen / Q000494:pharmacology; D011489:Protein Denaturation; D012643:Selenium / Q000032:analysis; D013050:Spectrometry, Fluorescence
D001386:Azides; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D012643:Selenium; D003545:Cysteine; D010100:Oxygen