PRE-STEADY-STATE MGATP-DEPENDENT PROTON PRODUCTION AND ELECTRON-TRANSFER BY NITROGENASE FROM AZOTOBACTER-VINELANDII

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinedandii was studied by monitoring the absorbance chang es at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly b uffered solution. The absorbance changes are characterized by a consta nt phase, a single exponential decrease and a linear decrease. The obs erved rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 degrees C is 14+/-4 s(-1). No proton production with a rate constant comparable to the ob served rate constant of electron transfer (k(obs) approximate to 100 s (-1)) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase protein s before mixing with MgATP; less protons are produced when more electr ons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +/- 0.3 mol H-produced(+)/mol MoFe protein with oxidized Fe prote in, and 1.1 +/- 0.1 mol H-produced(+)/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons ar e taken up after electron transfer from the Fe protein to the MoFe pro tein; the ratio electrons(transferred)/H-uptake(+) was calculated to b e 1.2+/-0.2. After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not ha ve a constant phase and the observed rate constant of the single expon ential reaction is higher, compared to MgATP-dependent proton producti on (k(obs)approximate to 35 s(-1)). The amount of protons produced dep ends also on the redox state of the Fe protein; no proton production w as observed with the oxidized Fe protein; with dithionite-reduced Fe p rotein a value of 3.1 +/- 0.4 mol H-produced(+)/mol MoFe protein was f ound (or 0.5 +/- 0.1 mol H+/mol Fe protein). Similar results were obta ined when only the Fe protein was mixed with MgADP, but the observed a bsorbance changes were smaller; mixing of dithionite-reduced Fe protei n with MgADP resulted in the production of 0.17 +/- 0.05 mol H+/mol Fe protein. All reported absorbance changes were absent when the experim ents were performed in a buffered solution. The series of events that occur after mixing of the nitrogenase proteins with MgATP will be pres ented and discussed. In the case of the reduced Fe protein, electron t ransfer takes place at a rate of 100 s(-1), which is followed by H+ pr oduction (k(obs)approximate to 14 s(-1)). When there is no electron tr ansfer (oxidized Fe protein) the rate constant of the MgATP-induced pr oton production decreases. When electrons are transferred, stoichiomet rically less protons are produced.