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 v ≈ 13 and 8 MHz that can be assigned, respectively, to “distant ENDOR” from 13C in natural-abundance (1.1%) and from 14N (the ΔmI = ±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 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the π carbons of cysteines bound to the cluster [A(13C) ≈ 1.0 MHz]. Five proteins show resolved, local ΔmI = ±2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 ≲ A(14N) ≲ 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(14N) ≈ 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N-and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of ΔmI = ±2 signals at Q-band provides a new technique for examining weak interactions with a cluster. Six proteins show an additional pattern near v ≈ 18 MHz that arises from 57Fe in natural abundance (2.2%) with A(57Fe) ≈ 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 2H-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.
ASJC Scopus subject areas