Mössbauer and Integer-Spin EPR Studies and Spin-Coupling Analysis of the [4Fe-4S]0Cluster of the Fe Protein fromAzotobacter vinelandiiNitrogenase

Abstract

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 Mössbauer 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 δ = 0.68 mm/s. One site, FeA, has ΔEQ = 3.08 mm/s while the three other sites exhibit ΔEQ-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 α2 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, FeA, 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 Mössbauer spectroscopy we obtained for the S = 4 multiplet the zero-field splitting parameter D = −0.75 ± 0.05 cm-1. Moreover, the Mössbauer studies show that the two lowest levels of the spin multiplet are split by Δground = 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 (J) 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 aiso = −22.4 MHz of ferrous rubredoxin, yield Aiso-values for three of the sites that are in good agreement with the experimental data; FeA, however, requires a much smaller aiso-value (−12 to −16 MHz).