期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
卷 1837, 期 2, 页码 277-286出版社
ELSEVIER
DOI: 10.1016/j.bbabio.2013.10.013
关键词
Molybdenum; Nitrate reductase; Pyranopterin; Electron transfer; Protein film voltammetry; EPR spectroscopy
资金
- CNRS
- CEA
- Aix-Marseille Universite
- Agence Nationale de la Recherche (ANR MC2) [11-BSV5-005-01]
In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the high g species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) high g resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe-4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin-spin interactions between the Mo(V) ion and the [4Fe-4S](1+) cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe-4S](1+) centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe-4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form. (C) 2013 Elsevier B.V. All rights reserved.
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