Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa.
1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7 x 10(5) s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c-->d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
A
A
Bellelli
P
P
Brzezinski
M
M
Arese
F
F
Cutruzzola
M C
MC
Silvestrini
M
M
Brunori
D004579:Electron Transport; D009572:Nitrite Reductases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D011994:Recombinant Proteins; D009572:Nitrite Reductases; D015032:Zinc