Binding of Histidine in the (Cys)3(His)1-Coordinated [2Fe−2S] Cluster of Human mitoNEETy

摘要

Abstract: Human mitoNEET is a homodimeric iron sulfur protein located in the outer mitochondrialnmembrane with unknown function, but which is known to interact with thiazolidinedione diabetes drugs.nEach monomer houses a [2Fe 2S] cluster with an unusual (Cys)3(His)1 ligation. The His ligand is importantnfor enabling cluster release and for tuning the redox potential. We use multifrequency (X-, Ka-, and Q-band)nand multitechnique (continuous-wave, electron spin-echo envelope modulation (ESEEM), pulsednelectron nuclear double resonance (ENDOR), and hyperfine sublevel correlation (HYSCORE)) electronnparamagnetic resonance spectroscopy to investigate the cluster in its paramagnetic reduced [Fe2 Fe3 ]n(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 fallsnbetween those of Rieske-type and ferredoxin-type [2Fe 2S] clusters. Simulation and least-squares fittingnof orientation-selective Ka- and Q-band ENDOR, 1D ESEEM, and HYSCORE spectra ofn14N and 15N-nlabeled mitoNEET yield the principal values and orientations of both the hyperfine tensor (n14N, Aiso 6.25nMHz, T 0.94 MHz) and the quadrupolar tensor (e2Qq/h 2.47 MHz, η 0.38) of the ligating histidinennitrogen Nδ. From these, we can infer the absolute g tensor orientation with respect to the cluster: The g2naxis is close to perpendicular to the [2Fe 2S] plane, and g1 and g3 are in-plane, but skewed from thenFe Fe and S S axes. In X-band ENDOR and ESEEM spectra, a weakly coupled nitrogen is visible, mostnlikely the Nε of the histidine in the protonated state. We find that the cluster is in a valence-localized state,nwhere Fe2 is His-bound. The field-sweep spectra show evidence of intercluster dipolar coupling that cannbe simulated using an uncoupled spin model for each cluster (SFe2 2, SFe3 5/2). The parametersndetermined in this work can function as reporters on how the cluster structure is altered upon pH changesnand drug binding.