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1.
Biochemistry ; 60(31): 2419-2424, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34310123

RESUMO

The human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron-sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer, and possibly Parkinson's disease. The geometric structure of mNT as a function of redox state and pH is critical for its function. In this study, we combine 57Fe nuclear resonance vibrational spectroscopy with density functional theory calculations to understand the novel properties of this important protein.


Assuntos
Cisteína/química , Ferro/química , Lisina/química , Proteínas Mitocondriais/química , Enxofre/química , Teoria da Densidade Funcional , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Vibração
2.
J Am Chem Soc ; 143(22): 8237-8243, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34043346

RESUMO

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe]H subcluster. A -13C2H2- ADT labeling in the synthetic diiron precursor of [2Fe]H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), and NRVS was measured on samples poised in the catalytically crucial Hhyd state containing a terminal hydride at the distal Fe site. The 13C2H isotope effects were observed also in the Hhyd spectrum. DFT simulations of the spectra allowed identification of the 57Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H2 formation.


Assuntos
Hidrogênio/metabolismo , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Isótopos de Carbono , Teoria da Densidade Funcional , Deutério , Hidrogênio/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Marcação por Isótopo , Conformação Molecular , Vibração
3.
Chem Sci ; 11(21): 5487-5493, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34094075

RESUMO

A diiron complex containing a bridging hydride and a protonated terminal thiolate of the form [(µ,κ2-bdtH)(µ-PPh2)(µ-H)Fe2(CO)5]+ has been investigated through 57Fe nuclear resonance vibrational spectroscopy (NRVS) and interpreted using density functional theory (DFT) calculations. We report the Fe-µH-Fe wagging mode, and indications for Fe-µD stretching vibrations in the D-isotopologue, observed by 57Fe-NRVS. Our combined approach demonstrates an asymmetric sharing of the hydride between the two iron sites that yields two nondegenerate Fe-µH/D stretching vibrations. The studied complex provides an important model relevant to biological hydrogen catalysis intermediates. The complex mimics proposals for the binuclear metal sites in [FeFe] and [NiFe] hydrogenases. It is also an appealing prototype for the 'Janus intermediate' of nitrogenase, which has been proposed to contain two bridging Fe-H-Fe hydrides and two protonated sulfurs at the FeMo-cofactor. The significance of observing indirect effects of the bridging hydride, as well as obstacles in its direct observation, is discussed in the context of biological hydrogen intermediates.

4.
J Am Chem Soc ; 142(1): 222-232, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31820961

RESUMO

[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.


Assuntos
Monóxido de Carbono/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Chlamydomonas reinhardtii/metabolismo , Teoria da Densidade Funcional , Desulfovibrio desulfuricans/metabolismo , Transporte de Elétrons , Ressonância Magnética Nuclear Biomolecular/métodos
5.
Chem Sci ; 10(32): 7535-7541, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31588304

RESUMO

Iron-sulfur clusters are common building blocks for electron transport and active sites of metalloproteins. Their comprehensive investigation is crucial for understanding these enzymes, which play important roles in modern biomimetic catalysis and biotechnology applications. We address this issue by utilizing (Et4N)3[Fe4Te4(SPh)4], a tellurium modified version of a conventional reduced [4Fe-4S]+ cluster, and performed both 57Fe- and 125Te-NRVS to reveal its characteristic vibrational features. Our analysis exposed major differences in the resulting 57Fe spectrum profile as compared to that of the respective [4Fe-4S] cluster, and between the 57Fe and 125Te profiles. DFT calculations are applied to rationalize structural, electronic, vibrational, and redox-dependent properties of the [4Fe-4Te]+ core. We herein highlight the potential of sulfur/tellurium exchange as a method to isolate the iron-only motion in enzymatic systems.

6.
Angew Chem Int Ed Engl ; 57(33): 10605-10609, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-29923293

RESUMO

A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We observed a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.


Assuntos
Hidrogenase/química , Domínio Catalítico , Clostridium/enzimologia , Teoria da Densidade Funcional , Desulfovibrio desulfuricans/enzimologia , Hidrogenase/genética , Hidrogenase/metabolismo , Ferro/química , Mutagênese Sítio-Dirigida , Prótons , Espectroscopia de Infravermelho com Transformada de Fourier
7.
J Am Chem Soc ; 139(46): 16894-16902, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29054130

RESUMO

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior to H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.


Assuntos
Hidrogênio/metabolismo , Hidrogenase/metabolismo , Ferro/metabolismo , Teoria Quântica , Biocatálise , Domínio Catalítico , Chlamydomonas reinhardtii/enzimologia , Desulfovibrio desulfuricans/enzimologia , Modelos Moleculares , Análise Espectral , Vibração
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