The iron (Fe) proteins of molybdenum (Mo)-, vanadium (V)-, and iron (Fe)-only nitrogenases are encoded by nifH, vnfH, and anfH, respectively. While the nifH-encoded Fe protein has been extensively studied over recent years, information regarding the properties of the vnfH- and anfH-encoded Fe proteins has remained scarce. Here, we present a combined biochemical, electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) analysis of the [Fe4S4] clusters of NifH, VnfH, and AnfH of Azotobacter vinelandii. Our data show that all three Fe proteins contain [Fe4S4] clusters of very similar spectroscopic and geometric structural properties, although NifH differs more from VnfH and AnfH with regard to the electronic structure. These observations have an interesting impact on the theory of the plausible sequence of evolution of nitrogenase Fe proteins. More importantly, the results presented herein provide a platform for future investigations of the differential activities of the three Fe proteins in nitrogenase biosynthesis and catalysis.