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Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center.

ABSTRACT: MitoNEET is a 2Fe-2S outer mitochondrial membrane protein that was initially identified as a target for anti-diabetic drugs. It exhibits a novel protein fold, and in contrast to other 2Fe-2S proteins such as Rieske proteins and ferredoxins, the metal clusters in the mitoNEET homodimer are each coordinated by one histidine residue and three cysteine residues. The interaction of the ligating His87 residue with the 2Fe-2S moiety is especially significant because previous studies have shown that replacement with Cys in the H87C mutant stabilizes the cluster against release. Here, we report the resonance Raman spectra of this naturally occurring Fe(2)S(2)(His)(Cys)(3) protein to assess local structural changes associated with cluster lability. Comparison of mitoNEET to its ferredoxin-like H87C mutant indicates that Raman peaks in the approximately 250-300 cm(-1) region of mitoNEET are influenced by the Fe-His87 moiety. Systematic pH-dependent resonance Raman spectral changes were observed in this spectral region for native mitoNEET but not the H87C mutant. The approximately 250-300 cm(-1) region of native mitoNEET is also sensitive to phosphate buffer. Thus, conditions that influence cluster release are shown here to concomitantly affect the resonance Raman spectrum in the region with Fe-His contribution. These results support the hypothesis that the Fe-N(His87) interaction is modulated within the physiological pH range, and this modulation may be critical to the function of mitoNEET.

SUBMITTER: Tirrell TF

PROVIDER: S-EPMC2861891 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center.

Tirrell Timothy F TF Paddock Mark L ML Conlan Andrea R AR Smoll Eric J EJ Nechushtai Rachel R Jennings Patricia A PA Kim Judy E JE

Biochemistry 20090601 22

MitoNEET is a 2Fe-2S outer mitochondrial membrane protein that was initially identified as a target for anti-diabetic drugs. It exhibits a novel protein fold, and in contrast to other 2Fe-2S proteins such as Rieske proteins and ferredoxins, the metal clusters in the mitoNEET homodimer are each coordinated by one histidine residue and three cysteine residues. The interaction of the ligating His87 residue with the 2Fe-2S moiety is especially significant because previous studies have shown that rep ...[more]

PMID: 19388667

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Project description: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.

Dataset Information

Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center.

Publications

Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center.

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