Conformational distribution in protein-bound [3Fe-4S]⁺ clusters: CW and pulsed EPR and ⁵⁷Fe ENDOR of D. gigas hydrogenase

This paper discusses 9- and 35-GHz EPR spectra of the Desulfovibrio gigas [3Fe-4S]⁺ cluster and also describes 35-GHz CW ⁵⁷Fe ENDOR measurements of isotopically enriched protein. The EPR spectra of [3Fe-4S]⁺ clusters typically cannot be described by a well-defined g tensor. Their properties instead can be modeled with a formal treatment that assumes there is a distribution in the g values. This treatment implicitly assumes that the EPR spectra of such clusters are controlled by a distribution in protein conformation and cluster structure, but the validity of this assumption has never been confirmed by an experiment that correlates the EPR spectrum of a cluster with a microscopic cluster property. We now report such a correlation. We find that the EPR and the ⁵⁷Fe ENDOR spectra for the D. gigas cluster can be described jointly in terms of a superposition of contributions from a bimodal distribution in cluster forms. Decomposition of the EPR spectra indicates that the major form has a well-defined structure as reflected in a well-defined g tensor with principal values 2.032, 2.024, and 2.016. The other, minority form shows a significant distribution in g tensor values. ⁵⁷Fe ENDOR measurements show that the iron ion of the cluster with the largest hyperfine coupling has quite different properties in the two forms, thus confirming the implicit assumption in decompositions of the EPR spectra that the protein exhibits different substates in which the cluster exhibits differing structures and properties.