Pre‐Steady‐State MgATP‐Dependent Proton Production and Electron Transfer by Nitrogenase from Azotobacter vinelandii

Abstract

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 "C is 14 2 4 s-'. No proton production with a rate constant comparable to the observed rate constant of electron transfer (/coba = 100 s-l) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +-0.3 mol H&duced/mOl MoFe protein with oxidized Fe protein, and 1.1 2 0.1 mol H&duced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electronsuansre,,ed~/H,+,,,, was calculated to be 1.2 2 0.2.After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (koba = 35 sP).The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein ; with dithionite-reduced Fe protein a value of 3.1 2 0.4 mol H&,duCed/mol MoFe protein was found (or 0.5 2 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 2 0.05 mol H+/mol Fe protein.All reported absorbance changes were absent when the experiments were performed in a buffered solution.The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-', which is followed by H' production (kobs = 14 ss'). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases. When electrons are transferred, stoichiometrically less protons are produced.Nitrogenase is the enzyme system which catalyses the reaction of nitrogen fixation, in which dinitrogen is reduced to ammonia. Nitrogenase consists of two distinct oxygensensitive metalloproteins, which are both necessary for catalytic activity [l -31. The molybdenum-containing nitrogenase was studied in the present investigation. The largest of the nitrogenase proteins is the MoFe protein (Avl); it contains two types of metal-sulphur clusters, an Fe-, Mo-and Scon...

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

“…The molar concentrations of Avl and Av2 were calculated from their molecular masses, 230 kDa and 63 kDa, respectively. Avl contained 1.8 + 0.2 Mo/mol Avl; the iron content of the Fe protein was 3.6 + 0.3 mol Fe/mol Av2 [7].…”

“…The acetylene reduction activity (at 30°C) of the nitrogenase proteins was determined as described elsewhere [7]. The specific activity of each of the nitrogenase proteins was calculated from the maximum acetylene reduction rate, which was obtained at an optimum Fig.…”

“…Pro-tein concentrations were estimated by the microbiuret method after a precipitation step with deoxycholic acid and trichloric acid [7]. The molar concentrations of Avl and Av2 were calculated from their molecular masses, 230 kDa and 63 kDa, respectively.…”

“…Azotobacter vinelandii ATCC strain 478 was grown and the nitrogenase component proteins were purified as described elsewhere [7]. Pro-tein concentrations were estimated by the microbiuret method after a precipitation step with deoxycholic acid and trichloric acid [7].…”

“…In the Fe protein cycle the reduced Fe protein (with MgATP bound) and the MoFe protein associate which yields the active nitrogenase complex. In the nitrogenase complex a single electron is transferred from the Fe protein to the MoFe protein and MgATP is hydrolysed [7]. Hereafter the nitrogenase complex dissociates into the separate nitrogenase proteins: this is the rate-limiting step of the cycle [8].…”