RESUMEN
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.
Asunto(s)
Cisteína , Histidina/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Azufre , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Hierro , Ligandos , Proteínas Mitocondriales/genética , Modelos Moleculares , Mutación , Nitrógeno , Conformación Proteica , Tiazolidinedionas/metabolismoRESUMEN
The Rieske protein from Thermus thermophilus (TtRp) and a truncated version of the protein (truncTtRp), produced to achieve a low-pH crystallization condition, have been characterized using UV-visible and circular dichroism spectroscopies. TtRp and truncTtRp undergo a change in the UV-visible spectra with increasing pH. The LMCT band at 458 nm shifts to 436 nm and increases in intensity. The increase at 436 nm versus pH can be fit using the sum of two Henderson-Hasselbalch equations, yielding two pK(a) values for the oxidized protein. For TtRp, pK(ox1) = 7.48 +/- 0.12 and pK(ox2) = 10.07 +/- 0.17. For truncTtRp, pK(ox1) = 7.87 +/- 0.17 and pK(ox2) = 9.84 +/- 0.42. The shift to shorter wavelength and the increase in intensity for the LMCT band with increasing pH are consistent with deprotonation of the histidine ligands. A pH titration of truncTtRp monitored by circular dichroism also showed pH-dependent changes at 315 and 340 nm. At 340 nm, the fit gives pK(ox1) = 7.14 +/- 0.26 and pK(ox2) = 9.32 +/- 0.36. The change at 315 nm is best fit for a single deprotonation event, giving pK(ox1) = 7.82 +/- 0.10. The lower wavelength region of the CD spectra was unaffected by pH, indicating that the overall fold of the protein remains unchanged, which is consistent with crystallographic results of truncTtRp. The structure of truncTtRp crystallized at pH 6.2 is very similar to TtRp at pH 8.5 and contains only subtle changes localized at the [2Fe-2S] cluster. These titration and structural results further elucidate the histidine ligand characteristics and are consistent with important roles for these amino acids.
Asunto(s)
Proteínas Bacterianas/química , Complejo III de Transporte de Electrones/química , Concentración de Iones de Hidrógeno , Thermus thermophilus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Transporte de Electrón , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Cinética , Modelos Moleculares , Conformación Proteica , Espectrofotometría , Thermus thermophilus/genéticaRESUMEN
The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.