A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase.
Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.
D055598:Chemical Phenomena; D002621:Chemistry; D004120:Dimethyl Suberimidate; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D009566:Nitrates; D009569:Nitric Oxide; D009572:Nitrite Reductases / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013053:Spectrophotometry
D009566:Nitrates; D004120:Dimethyl Suberimidate; D009569:Nitric Oxide; D007501:Iron; D009572:Nitrite Reductases; D003576:Electron Transport Complex IV