EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

González, Pablo J.; Rivas, Maria G.; Brondino, Carlos D.; Bursakov, Sergey A.; Moura, Isabel; Moura, José J. G.

doi:10.1007/s00775-006-0110-0

Cited by 40 publications

(63 citation statements)

References 40 publications

(63 reference statements)

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“…Our analysis of the E. coli NapA Mo 5ϩ EPR signal also reveals that it is very similar to a signal recently described for the monomeric D. desulfuricans NapA (43). Like that of E. coli NapA and NapAB, this signal is not present in 'as prepared' enzyme but it can be resolved in a stable state following reduction of the enzyme with dithionite and treatment with nitrate ( Fig.…”

Section: Figure 3 Spectropotentiometric Properties Of the Mosupporting

confidence: 85%

“…The description of this type of signal presented here for the E. coli NapA in the absence of nitrate confirms that this Mo 5ϩ nitrate signal is not a nitrate-bound form. However, given the failure to identify a High g Resting signal, it does add weight the view that it could be a catalytically relevant signal, and it is compelling to note that a very similar signal has now also been described during turnover of D. desulfuricans NapA (43,44) (Table 3 [Turnover]).…”

Section: Figure 3 Spectropotentiometric Properties Of the Momentioning

confidence: 99%

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Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Jepson¹,

Mohan

Clarke³

et al. 2007

Journal of Biological Chemistry

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The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems of ␣-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the ␥-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 M determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the ␣-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 Å , which is indicative of a water ligand. The potential range over which the Mo 6؉ state is reduced to the Mo 5؉ state in either NapA (between ؉100 and ؊100 mV) or the NapAB complex (؊150 to ؊350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo 6؉/5؉ > ؉350 mV), and the form of the Mo 5؉ EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo 5؉ state could not be further reduced to Mo 4؉. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo 5؉ ion and where a stable des-oxo Mo 6؉ species may participate.Bacterial nitrate reductases are molybdoenzymes that catalyze the two-electron reduction of nitrate to nitrite. They can be classified into three groups according to their localization and function, namely membrane-bound respiratory, periplasmic respiratory, or cytoplasmic assimilatory enzymes (1, 2). Bacterial respiratory membrane-bound nitrate reductases, such as Escherichia coli NarGHI, are generally integral membrane protein complexes that have an active site on the cytoplasmic face of the membrane and couple quinol oxidation by nitrate to the generation of a transmembrane proton electrochemical gradient (2). The catalytic subunit, NarG, contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) 3 cofactor and a [4Fe-4S] cluster (3, 4). Periplasmic nitrate reductases (Nap) are also linked to quinol oxidation in respiratory electron transport chains, but do not conserve the free energy of the QH 2 -nitrate couple. Nitrate reduction via Nap can be coupled to energy conservation if the primary quinone reductase, for example NADH dehydrogenase or formate dehydrogenase, generates a proton electrochemical gradient. Thus Nap systems have a range of physiological functions that include the disposal of reducing equivalents during aerobic growth on reduced carbon substrates and anaerob...

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Section: Figure 3 Spectropotentiometric Properties Of the Mosupporting

confidence: 85%

Section: Figure 3 Spectropotentiometric Properties Of the Momentioning

confidence: 99%

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Jepson¹,

Mohan

Clarke³

et al. 2007

Journal of Biological Chemistry

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“…EPR studies show different types of signals under different experimental conditions [9]. Reduction with dithionite of as-prepared enzyme yields a rhombic EPR signal called ''low-potential signal'' (g values 2.016, 1.987, 1.964), which is replaced by the nitrate signal (g values 2.000, 1.990, 1.981) upon nitrate addition.…”

Section: Introductionmentioning

confidence: 99%

“…This variability of the electronic properties of the molybdenum active site must be necessarily reflected in its structural properties, which suggests that the active site of these enzymes presents a high flexibility of coordination. Although tentative structures of the EPR-active species in Nap have been proposed [9,10], a clear correlation between structure and EPR properties has not been clearly established yet and requires further investigation.…”

Section: Introductionmentioning

confidence: 99%

“…These studies indicate that the binding of the nitrate molecule to the molybdenum atom occurs not only in the Mo(IV) form, but also in the Mo(V) form, thus supporting a reaction scheme which can also occur through two sequential steps of one electron in parallel to the single two-electron step as discussed above. More recently, EPR studies on DdNapA in catalytic conditions detected a novel EPR signal called ''turnover'' (g values 1.999, 1.990, 1.982) which had not been detected for any Nap before [9]. Although these studies were not conclusive regarding the nature of the turnover species, they suggested that the ready state of the enzyme to initiate the reaction might also be the Mo(V) redox state, which is in line with the electrochemical studies performed on RsNapAB, EcNapA, and SeNarB [6,12,13].…”

Section: Introductionmentioning

confidence: 99%

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Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

et al. 2008

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Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 Å . The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH 2 ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (k = 1.77 Å ) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sc atom of the molybdenum ligand Cys A140 was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 Å , indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with 15 N-labeled nitrate and in D 2 O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligandbased redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

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Vacancy Augmented Piezo‐Sonosensitizer for Cancer Therapy

Zhang

Pan

et al. 2023

Advanced Science

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Sonodynamic therapy (SDT) has been widely reported as a noninvasive and high‐penetration therapy for cancer; however, the design of an efficient sonosensitizer remains an urgent need. To address this issue, molybdenum disulfide nanoflowers (MoS2 NF) as piezo‐sonosensitizers and introduced sulfur vacancies on the MoS2 NF (Sv‐MoS2 NF) to improve their piezoelectric property for cancer therapy are designed. Under ultrasonic mechanical stress, the Sv‐MoS2 NF resulted in piezoelectric polarization and band tilting, which enhanced the charge carrier separation and migration. This resulted in an improved catalytic reaction for reactive oxygen species (ROS) production, ultimately enhancing the SDT performance. Thanks to the high efficiency of ROS generation, the Sv‐MoS2 NF have demonstrated a good anticancer effect in vitro and in vivo. Following a systematic evaluation, Sv‐MoS2 NF also demonstrated good biocompatibility. This novel piezo‐sonosensitizer and vacancy engineering strategy provides a promising new approach for achieving efficient SDT.

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EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

Cited by 40 publications

References 40 publications

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Vacancy Augmented Piezo‐Sonosensitizer for Cancer Therapy

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