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 [Fe(2+)Fe(3+)] (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 (14)N and (15)N-labeled mitoNEET yield the principal values and orientations of both the hyperfine tensor ((14)N, A(iso) = -6.25 MHz, T = -0.94 MHz) and the quadrupolar tensor (e(2)Qq/h = -2.47 MHz, eta = 0.38) of the ligating histidine nitrogen N(delta). From these, we can infer the absolute g tensor orientation with respect to the cluster: The g(2) axis is close to perpendicular to the [2Fe-2S] plane, and g(1) and g(3) 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 N(epsilon) of the histidine in the protonated state. We find that the cluster is in a valence-localized state, where Fe(2+) 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(Fe(2+)) = 2, S(Fe(3+)) = 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.
Dicus
Michelle M
MM
Department of Chemistry, University of California, Davis, California 95616, USA.
Conlan
Andrea
A
Nechushtai
Rachel
R
Jennings
Patricia A
PA
Paddock
Mark L
ML
Britt
R David
RD
Stoll
Stefan
S
D001665:Binding Sites; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007501:Iron; D008024:Ligands; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009584:Nitrogen; D011487:Protein Conformation; D013455:Sulfur; D045162:Thiazolidinediones / Q000378:metabolism
C525595:CISD1 protein, human; D008024:Ligands; D024101:Mitochondrial Proteins; D045162:Thiazolidinediones; D006639:Histidine; D013455:Sulfur; C089946:2,4-thiazolidinedione; D007501:Iron; D003545:Cysteine; D009584:Nitrogen