Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S] + synthetic analog clusters

Abstract

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S] + clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe 4S 4(SEt) 4] 3− and [Fe 4S 4(SC 6H 11) 4] 3−. The results indicate similar [4Fe-4S] + clusters with analogous S = 1 2 and S = 3 2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1 2 and S = 3 2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1 2 and S = 3 2 forms of the [4Fe-4S] + have been determined. Together with the VTMCD studies of [Fe 4S 4(SEt) 4] 3− and [Fe 4S 4(SC 6H 11) 4] 3−, which are shown to be predominantly S = 1 2 and 3 2 , respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1 2 and S = 3 2 [4Fe-4S] + clusters. Ground state zero-field splitting parameters for the S = 3 2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1 2 resonance. A rapidly relaxing axial resonance, g ⊥ = 1.94 and g∥ = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S] + clusters in frozen solution is discussed in light of these results.