14,15N, 13C, 57Fe, and 1,2H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands

Abstract

The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near nu almost-equal-to 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR' from C-13 in natural-abundance (1.1%) and from N-14 (the DELTA-m(I) = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local C-13 ENDOR signals with resolved hyperfine interactions; these are assigned to the beta-carbons of cysteines bound to the cluster [A(C-13) almost-equal-to 1.0 MHz]. Five proteins show resolved, local DELTA-m1 = +/- 2 ENDOR signals from N-14 with an isotropic hyperfine coupling, 0.4 less than or similar to A(N-14) less than or similar to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(N-14) almost-equal-to 4 MHz. Quadrupole coupling constants are derived for both local and distant N-14 signals. The interpretation of the data is supported by studies on N-15- and C-13-enriched ferredoxin (Fd) from Anabaena 7120, where the N-15 signals can be clearly correlated with the corresponding N-14 signals and where the C-13 Signals are strongly enhanced. Thus, the observation of N-14 DELTA-m(I) = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster. Six proteins show an additional pattern near nu almost-equal-to 18 MHz that arises from Fe-57 in natural abundance (2.2%) with A(Fe-57) almost-equal-to 36 MHz, which opens the possibility of studying proteins for which enrichment is impractical. Q-band ENDOR studies also have been carried out on four H-2-exchanged Fe-S proteins, and ENDOR detects exchangeable protons in each. The importance of these findings for the interpretation of X- and Q-band ENDOR at low radiofrequencies is discussed.