PRE-STEADY-STATE KINETICS OF NITROGENASE FROM AZOTOBACTER-VINELANDII - EVIDENCE FOR AN ATP-INDUCED CONFORMATIONAL CHANGE OF THE NITROGENASECOMPLEX AS PART OF THE REACTION-MECHANISM

The pre-steady-state electron transfer reactions of nitrogenase from A zotobacter vinelandii have been studied by stopped-flow spectrophotome try. With reduced nitrogenase proteins after the initial absorbance in crease at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbanc e decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogena se proteins with MgATP leads after 20 ms to a decrease of the absorban ce, which could be fitted (from 0.05 to 1 s) with a single exponential decay with a rate constant k(obs)=6.6+/-0.8 s(-1). This reaction of n itrogenase was measured at different wavelengths. The data indicate th e formation of a species with a blue shift of the absorbance of metal- sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decr ease at 430 nm observed (after 50 ms) in the case of the reduced nitro genase proteins could only be simulated well if, after the initial ele ctron transfer from the Fe protein to the MoFe protein and before diss ociation of the nitrogenase complex, an additional reaction was assume d. The rate constant of this reaction was of the same order as the rat e constant of the MgATP-dependent pre-steady-state proton production b y nitrogenase from A. vinelandii: k(obs)=14+/-4 s(-1) with reduced nit rogenase proteins and k(obs)=6+/-2 s(-1) with dithionite-free nitrogen ase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J . Biochem, 225, 881-890). It is proposed that in the presence and abse nce of reductant, the observed absorbance decrease at 430 nm of nitrog enase is caused by a change of the conformation of the nitrogenase com plex, as a consequence of hydrolysis of MgATP.