Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by 57Fe Mössbauer spectroscopy and by Fe and Zn EXAFS as well as XANES spectroscopy. The analyses were performed on the basis of data known for the FeMo cofactor and the P cluster of Mo nitrogenases. The prominent Fourier transform peaks of the Fe K-edge spectrum are assigned to Fe-S and Fe-Fe interactions at distances of 2.29 A and 2.63 A, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 A, which is an unprecedented feature of the trigonal prismatic arrangement of iron atoms found in the FeMo cofactor of nitrogenase MoFe protein crystal structures. Additional Fe...Fe interactions at 2.92 A and 4.05 A clearly indicate that the principal geometry of the P cluster is also conserved. Mössbauer spectra of 57Fe-enriched FeFe protein preparations were recorded at 77 K (20 mT) and 4.2 K (20 mT, 6.2 T), whereby the 4.2 K high-field spectrum clearly demonstrates that the cofactor of the Fe-only nitrogenase (FeFe cofactor) is diamagnetic in the dithionite-reduced ("as isolated") state. The evaluation of the 77 K spectrum is in agreement with the assumption that this cofactor contains eight Fe atoms. In the literature, several genetic and biochemical lines of evidence are presented pointing to a significant structural similarity of the FeFe, the FeMo and and the FeV cofactors. The data reported here provide the first spectroscopic evidence for a structural homology of the FeFe cofactor to the heterometal-containing cofactors, thus substantiating that the FeFe cofactor is the largest iron-sulfur cluster so far found in nature.

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In the presence of molybdate (1 μM) 2–3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen‐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 ethylene reduced ċ min−1· mg protein−1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible 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. However, the molybdate‐containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases. The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non‐saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH‐dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S =3/2 FeMo cofactor ESR signal was 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 intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.

One of the primary challenges of chemistry is the controlled synthesis of compounds with tailor‐made structures and properties. Natural products serve as inspiration in this quest, ranging from biocatalysts with optimal selectivity and activity to “inorganic materials” with exceptional properties, whose generation can be described by the term biomineralization. It is of fundamental importance to comprehend the courses of events at the interface between gene expression and the subsequent processes of epigenesis that are no longer under gene control. Chemistry has been able to achieve many goals; however, in the area of controlled syntheses of highly complex, tailor‐made metal clusters, there is a lack of fundamental theories and principles. This is especially true for the fascinating metal–sulfur cluster of nitrogenase, which, in this enzyme, functions as the active center for the N2 reduction and, so far, has eluded all attempts to be synthesized in the laboratory. To understand the biosynthesis of this cluster, information from genetics and chemistry must be combined.