Binding of histidine in the (Cys)3(His)1-coordinated [2Fe-2S] cluster of human mitoNEET

Michelle M. Dicus, Andrea Conlan, Rachel Nechushtai, Patricia A. Jennings, Mark L. Paddock, R. David Britt, Stefan Stoll

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Human mitoNEET is a homodimeric iron-sulfur protein located in the outer mitochondrial membrane with unknown function, but which is known to interact with thiazolidinedione diabetes drugs. Each monomer houses a [2Fe-2S] cluster with an unusual (Cys)3(His)1 ligation. The His ligand is important for enabling cluster release and for tuning the redox potential. We use multifrequency (X-, Ka-, and Q-band) and multitechnique (continuous-wave, electron spin-echo envelope modulation (ESEEM), pulsed electron-nuclear double resonance (ENDOR), and hyperfine sublevel correlation (HYSCORE)) electron paramagnetic resonance spectroscopy to investigate the cluster in its paramagnetic reduced [Fe2+Fe3+] (S ) 1/2) state. It has a rhombic g tensor (2.007, 1.937, 1.897) with an average g value of 1.947 that falls between those of Rieske-type and ferredoxin-type [2Fe-2S] clusters. Simulation and least-squares fitting of orientation-selective Ka- and Q-band ENDOR, 1D ESEEM, and HYSCORE spectra of 14N and 15N- labeled mitoNEET yield the principal values and orientations of both the hyperfine tensor (14N, Aiso)-6.25 MHz, T = -0.94 MHz) and the quadrupolar tensor (e2Qq/h)-2.47 MHz,η = 0.38) of the ligating histidine nitrogen Nδ. From these, we can infer the absolute g tensor orientation with respect to the cluster: The g2 axis is close to perpendicular to the [2Fe-2S] plane, and g1 and g3 are in-plane, but skewed from the Fe-Fe and S-S axes. In X-band ENDOR and ESEEM spectra, a weakly coupled nitrogen is visible, most likely the Nε of the histidine in the protonated state. We find that the cluster is in a valence-localized state, where Fe2+ is His-bound. The field-sweep spectra show evidence of intercluster dipolar coupling that can be simulated using an uncoupled spin model for each cluster (SFe2+ = 2, SFe3+ = 5/2). The parameters determined in this work can function as reporters on how the cluster structure is altered upon pH changes and drug binding.

Original languageAmerican English
Pages (from-to)2037-2049
Number of pages13
JournalJournal of the American Chemical Society
Issue number6
StatePublished - 2010


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