Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

Raleiras, Patrícia; Kellers, Petra; Lindblad, Peter; Magnuson, Ann

doi:10.1074/jbc.m113.468587

Cited by 14 publications

(34 citation statements)

References 49 publications

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“…3B, no absorption spectra of FMN were detected. The UV-visible absorption spectra of the as-purified and dithionite-reduced LldABC are consistent with the iron-sulfur clusters (42,43). The presence of the iron-sulfur cluster in LldABC was further identified by elemental analyses.…”

Section: Resultssupporting

confidence: 54%

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

Gao¹,

Wang²,

Zhang³

et al. 2015

J. Bacteriol.

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NAD-independent L-lactate dehydrogenases (L-iLDHs) play important roles in L-lactate utilization of different organisms. All of the previously reported L-iLDHs were flavoproteins that catalyze the oxidation of L-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of L-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive L-iLDH activity was detected. The expressed L-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified L-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified L-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing L-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the L-lactate oxidation. LldABC has narrow substrate specificity, and only L-lactate and DL-2-hydrobutyrate were rapidly oxidized. Mg 2؉ could activate L-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the L-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the L-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized L-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor. IMPORTANCEProviding new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent L-lactate dehydrogenase (L-iLDH) encoded by the gene cluster lldABC is indispensable for the L-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing L-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

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Section: Resultssupporting

confidence: 54%

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

Gao¹,

Wang²,

Zhang³

et al. 2015

J. Bacteriol.

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“…All hydrogen-oxidizing [17] (strain ATCC 29411/PCC 7524) and other cyanobacterial hydrogenases [18], however, are missing the bacterial histidine coordination and possess a glutamine at that position. Raleiras et al have characterized the iron-sulfur clusters of Nostoc punctiforme using electron paramagnetic resonance (EPR) spectroscopy [19].…”

Section: Distal Fes-cluster Modelmentioning

confidence: 99%

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

Petrenko

Stein

2017

Preprint

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Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron-sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from Density Functional Theory (DFT) calculations. The distal cubane FeS-cluster has a three-cysteine and one-histidine coordination [Fe 4 S 4 ](His)(Cys) 3 first ligation sphere. The reorganization energy (inner-and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and an all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe 4 S 4 ](Cys) 4 case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment, which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.

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“…The protein environment of the distal cluster is then not only a protectant of the redox active transition metal cluster but also a highly evolved electron transfer path maintaining an efficient ET. [19] (strain ATCC 29411 / PCC 7524) and other cyanobacterial hydrogenases [20], however, are missing the bacterial histidine coordination and possess a glutamine at that position. Raleiras et al have characterized the iron-sulfur clusters of Nostoc punctiforme using EPR spectroscopy [19].…”

Section: Distal Fes-clustermentioning

confidence: 99%

“…[19] (strain ATCC 29411 / PCC 7524) and other cyanobacterial hydrogenases [20], however, are missing the bacterial histidine coordination and possess a glutamine at that position. Raleiras et al have characterized the iron-sulfur clusters of Nostoc punctiforme using EPR spectroscopy [19]. From a characteristic EPR signature, they concluded the presence of a fully assembled, intact distal [Fe4S4]-cubane cluster with a His to Gln coordination but could not rationalize the effect of this mutation on redox potentials and ET rates.…”

Section: Distal Fes-clustermentioning

confidence: 99%

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

Petrenko¹,

Stein²

2017

Preprint

View full text Add to dashboard Cite

Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal ironsulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from DFT calculations. The distal cubane FeS-cluster has a three cysteine and one histidine coordination [Fe4S4](His)(Cys)3 first ligation sphere. The reorganization energy (inner-and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and the all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe4S4](Cys)4 case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.

show abstract

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

Cited by 14 publications

References 49 publications

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

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