[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster

Swanson, Kevin D.; Ratzloff, Michael W.; Mulder, David W.; Artz, Jacob H.; Ghose, Shourjo; Hoffman, Andrew O.; White, Spencer N.; Zadvornyy, Oleg A.; Broderick, Joan B.; Bothner, Brian; King, Paul W.; Peters, John W.

doi:10.1021/ja510169s

Cited by 127 publications

(168 citation statements)

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“…However, configurations with (semi) bridging or equatorial H-species were considered as well (12,14,36) and may result from structural flexibility of the H-cluster (36). Such structural dynamics may facilitate apical or equatorial ligand binding at the distal iron ion and may also be relevant for O 2 inactivation of the enzymes via reactive oxygen species formation (24,29,30,60,61). Our protocol for selective preparation of H ox with eight distinct isotopic labeling patterns introduces spectroscopic probes at individual positions at the cofactor.…”

Section: Discussionmentioning

confidence: 99%

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Senger

Mebs

Duan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

137

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The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H 2 -forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN − ) ligands in the active oxidized state (H ox ) and one additional CO ligand in the inhibited state (H ox -CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fouriertransform infrared spectroscopy. Combination of 13 CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 H ox and 16 H ox -CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For H ox -CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN − at the distal iron ion of the cofactor as opposed to an apical CO. For H ox , two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13 CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.[FeFe]-hydrogenase | isotope editing | infrared spectroscopy | density functional theory | cofactor dynamics T he reduction of protons to form molecular hydrogen (H 2 ) is catalyzed by [FeFe]-hydrogenases (1, 2). With a turnover rate of up to 10,000 H 2 molecules per second in a thermodynamically reversible reaction (3-5), [FeFe]-hydrogenases inspired synthetic hydrogen catalysts (6-8) and renewable fuel technology applications (9, 10). The mechanism of catalysis at the active site cofactor (H-cluster) needs to be elucidated. Further information on functional intermediates is required (11-16) and expected to emerge from spectroscopic studies on H-cluster constructs carrying siteselective isotopic reporter groups (17)(18)(19)(20) Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).The vibrational modes of the CO and CN − ligands at the diiron site are well accessible by infrared (IR) spectroscopy because they are separated from protein backbone and liquid water bands.Infrared spectroscopy therefore has pioneered elucidation of the molecular structure of the H-cluster and identification of several redox states (24, 31). In particular, the CO stretching frequencies are highly sensitive to structural isomerism, redox transitions, ligand binding, and isotope exchange (11,12,15,18,24,31,32). 13 CO editing ...

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Section: Discussionmentioning

confidence: 99%

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Senger

Mebs

Duan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

137

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“…However, this mechanism conflicts with a recent report according to which the O 2 -damaged enzyme from Cr harbors an intact 4Fe4S subsite and no 2Fe subcluster, and the observation that the O 2 -damaged enzyme is repaired upon insertion of a synthetic analogue of the 2Fe subcluster. 22 …”

Section: Introductionmentioning

confidence: 99%

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

et al. 2016

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FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of the highly reactive OH radical and hydroxylated cysteine, consistent with recent crystallographic evidence. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species at prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

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“…Inactivation of [Fe-Fe] hydrogenase on exposure to O 2 undergoes several steps. Under aerobic conditions O 2 enters into the protein structure and binds to enzyme catalytic site through gas migration channels, generating reactive oxygen species4567. This imparts irreversible oxidative damage of electron transfer domains and active site.…”

Section: Discussionmentioning

confidence: 99%

“…However, the oxygen (O 2 ) sensitivity of hydrogenase is a limitation for some practical applications4. Studies indicate that O 2 enters to the enzyme active site through the gas migration channels and on binding to the [Fe] moiety at the active site, generates reactive O 2 species that destroys the enzyme catalytic function567.…”

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confidence: 99%

Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

Mangayil

Karp

Lamminmäki

et al. 2016

Sci Rep

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Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

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[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster

Cited by 127 publications

References 50 publications

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

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