Escherichia coli Hmp, an "oxygen-binding flavohaemoprotein", produces superoxide anion and self-destructs.
Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) M(-1) s(-1) and k(off) 4.49x10(3) s(-1). The dissociation constant of the primary O(2) compound was 160 microM (25 degrees C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K(m) <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.
Wu
Guanghui
G
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
Corker
Hazel
H
Orii
Yutaka
Y
Poole
Robert K
RK
D001426:Bacterial Proteins / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology / Q000378:metabolism; D004093:Dihydropteridine Reductase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006420:Hemeproteins; C086193:flavohemoprotein, Bacteria; D009243:NAD; D013481:Superoxides; D009569:Nitric Oxide; D002248:Carbon Monoxide; D007501:Iron; D006735:Horseradish Peroxidase; D002374:Catalase; D013482:Superoxide Dismutase; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen