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 d(1)-haem; this r eaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d (1)-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d(1)-haem. 2. Photoexcitation of Zn-NIR is follow ed by electron transfer from the triplet excited state of the Zn-porph yrin 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 popula ted, we conclude that the back reaction is probably as fast. 3. Even t aking into account that in the native NIR the driving force is close t o zero, the rate constant for the c --> d(1) electron transfer, estima ted from our experiments, is still much higher than that observed by s topped flow (k = 0.3 s(-1)) using reduced azurin as the electron donor . This finding may be a direct kinetic indication that reduction of th e d(1)-haem is associated with a substantial reorganization of the co- ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.