Regulation of Nitrogen Fixation by Fe‐S Protein II in Azotobacter vinelandii

Abstract

1. Several low‐potential electron carriers from different sources can be photoreduced by the system 3,10‐dimethyl‐5‐deazaisoalloxazine/N‐tris(hydroxymethyl)methylglycine. The carriers studied were flavodoxin, ferredoxin I and iron‐sulfur protein II from Azotobacter vinelandii and the flavodoxins from Desulfovibrio vulgaris and Peptostreptococcus elsdenii. 2. Electron transport to A. vinelandii nitrogenase was studied, employing different preparations of the enzyme: a crude complex; a complex reconstituted from the 0.27 M and 0.38 M NaCl fractions after DEAE‐cellulose chromatography; a complex reconstituted from the 0.27 M and 0.38 M NaCl fraction plus iron‐sulfur protein II purified from the 0.15 M NaCl fraction. Of all photoreduced carriers tested, only flavodoxin hydroquinone from A. vinelandii catalyzes significant electron transport to these complexes. 3. The time course of oxidation of substrate‐amounts of A. vinelandii flavodoxin hydroquinone by catalytic amounts of crude nitrogenase complex shows three characteristic phases: an initial lag phase (1), a phase with constant rate over a range of redox potentials (2) and a final phase with rapidly declining rate (3). It was shown that the Fe‐S protein II is responsible for the lag phase; the potential where phase 2 changes into phase 3 is at a higher value in the presence of Fe‐S protein II. Pre‐reduction of the enzyme photochemically abolishes phase 1 and causes phase 2 to proceed at a higher rate. The rate in phase 2 can be enhanced also by lowering the ‘starting potential’ of the flavodoxin hydroquinone/semiquinone couple. 4. A. vinelandii flavodoxin shuttles between its hydroquinone and semiquinone forms during steady‐state electron transfer. Over 90% of the reducing equivalents is recovered in ethylene formed from acetylene. The concentrations of flavodoxin hydroquinone to give half‐maximum rate in the acetylene reduction assay is 3–6 μM. A scheme is proposed for the regulation of electron donation to nitrogenase. Copyright © 1977, Wiley Blackwell. All rights reserved