An analysis of g strain in the EPR of two [2Fe---2S] ferredoxins. Evidence for a protein rigidity model

Abstract

Replacing current notions of a paramagnetic center in a metalloprotein as a single entity in vivo with the more realistic concept of an ensemble of spin systems, each uniquely disturbed by its own surrounding protein, leads to a rigorous description of the spectroscopic factor, g, as a random variable whose statistical properties contain information on the rigidity of the protein. Generation of a consistent set of accurate simulations of very low-noise, multifrvquency (3, 9, 15 GHz) EPR data from selected proteins has now been achieved. This consistency lends support to the physical and biological inferences drawn from such simulations. The spectral contribution of magnetic hyperfine line-broadening is minimized by studying the 56Fe reconstituted [2Fe---2S] cluster in fully deuterated ferredoxin from Synechococcus lividus and the 2H2O exchanged [2Fe---2S] ferredoxin from Pseudomonas putida. High-resolution Mossbauer data on oxidized and reduced 57Fe reconstituted S. lividus ferredoxin are also presented. The oxidized spectrum shows that the inequivalence of the two ferric ions in a [2Fe---2S] cluster can be resolved as two Mossbauer lines. The complete absence of this splitting in the ferric fines of the reduced spectrum is definitive proof that the reducing electron always resides at the same 56Fe atom in frozen aqueous solutions. To explain the distributed nature of the paramagnetic site in the ferredoxins, three models are considered: (1) a multiplicity of EPR states; (2) external perturbations to the molecular Hamiltonian; (3) a distribution in the crystal field Hamiltonian parameters. The first model is discarded, the second is possible but difficult to verify, and the third model is shown to fit the data well. The latter comparison requires a correction to literature expressions for the g and A tensors in [2Fe---2S] clusters. Statistical analysis strongly suggests that the EPR of metalloproteins in its details is a reflection of protein structure that distributes its spatial coordinates, accommodating different levels of rigidity, the more flexible parts being located at the outside.