THE MOLYBDENUM NITROGENASE FROM WILD-TYPE XANTHOBACTER-AUTOTROPHICUS EXHIBITS PROPERTIES REMINISCENT OF ALTERNATIVE NITROGENASES

In the presence of molybdate (1 mu M) 2-3.5% oxygen and with sucrose a s carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic ni trogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm). The maximal specific activity obtained was 145 nmol ethy lene reduced . min(-1) . mg protein(-1) (crude extract). The Mo nitrog enase was derepressed to a comparable level with methionine as nitroge n source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenas e activity decreased to an extremely low level, The nitrogenase, respo nsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo-independent nitrogenase does not exist. How ever, the molybdate-containing nitrogenase exhibited some properties w hich are reminiscent of alternative nitrogenases. The MoFe protein (co mponent 1, Xa1) copurified with two molecules of a small, not previous ly detected polypeptide (molar mass 13.6 kDa) and was able to reduce a cetylene not only to ethylene but also partly ro ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titan ium (III) citrate as electron donor, at high component 1/component 2 r atios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH-dependent increase of the rel ative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal w as split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The inten sities of the two new FeMo cofactor signals were more pronounced the m ore alkaline the pH. They could be further enhanced using titanium (II I) citrate instead of Na2S2O4 as reductant.