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

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, A_iso)-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 (S_Fe²⁺ = 2, S_Fe³⁺ = 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.