We have previously shown that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to the all-ferrous state, [4Fe-4S](0). We have studied here this state with integer-spin EPR and Mossbauer spectroscopy, and analyzed the exchange couplings that give rise to a ground state with cluster spin S = 4. The results are as follows: The cluster contains four high-spin (Si = 2) ferrous sites with isomer shift delta = 0.68 mm/s. One site, Fe-A, has Delta E-Q = 3.08 mm/s while the three other sites exhibit Delta E-Q-values between 1.2 and 1.7 mm/s. This asymmetry is surprising in view of the fact that the cluster is suspended at the interface of an alpha(2) protein dimer. A similar 3:1 symmetry is also evident in the magnetic hyperfine tensors, A(i), of the four sites; three sites have negative A-values while that of the fourth site, Fe-A, is positive. Analysis of the exchange couplings, assumed to be antiferromagnetic, shows that every possible S = 4 ground multiplet that can be constructed from four high-spin ferrous sites must have this property. From Mossbauer spectroscopy we obtained for the S = 4 multiplet the zero-field splitting parameter D = -0.75 +/- 0.05 cm(-1) Moreover, the Mossbauer studies show that the two lowest levels of the spin multiplet are split by Delta(ground) = 0.02 cm(-1). Four pairs of levels from the S = 4 multiplet yield integer-spin EPR signals which can be observed, with only minor broadening, at temperatures up to 140 K. Transitions between the two lowest levels yield a sharp resonance at g = 16.4. We have simulated the EPR spectra of the all-ferrous cluster in the temperature range from 2 to 120 K and have obtained curves that fit the experimental data with high accuracy. The spin concentration obtained from these simulations is in good agreement with the [4Fe-4S] cluster concentration. Moreover, analysis of the EPR data has revealed that another spin multiplet, most likely an S = 3 manifold, becomes populated above 40 K. Exchange coupling (S) among four high-spin ferrous ions yields 85 spin multiplets, 15 of which have S = 4. To have an S = 4 ground state, two of the six J-values must differ by at least a factor 3, and two others by a factor 2.5. The computed spin projection factors, together with a(iso) = -22.4 MHz of ferrous rubredoxin, yield A(iso)-values for three of the sites that are in good agreement with the experimental data; Fe-A, however, requires a much smaller a(iso)-value (-12 to -16 MHz).