Cysteine 298 is one of the closest aminoacids to the Fed atom of the H-cluster of [FeFe]-hydrogenases. C298 was targeted in CaHydA by site saturation mutagenesis (SSM), a technique that randomly mutates the selected position into all the 20 aminoacids. The functional effects were simultaneously analysed with a specifically developed activity screening protocol, necessary to obtain quickly functional information from a large number of [FeFe]-hydrogenase variants. The screening protocol is a semi-quantitative colorimetric method that can be applied directly on E. coli colonies. Screening of the SSM library showed that the only amino acid that can replace cysteine with a remarkable activity is aspartic acid. Characterization of the purified C298D variant in comparison to the wild type revealed that the kcat decreases by 50% only and that there is no major structural effect. Interestingly, C298D causes a significant shift towards more acidic values of the pH activity profile. Characterization of the purified variants C298E, C298S, C298N and C298A, chosen because of structural similarity, further demonstrated that replacement of cysteine with other residues severely impairs the activity by 4 to 5 orders of magnitude or completely abolish it. The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and the shift in the pH activity profile demonstrate experimentally the central role of C298 in the proton transfer pathway to the active site during [FeFe]-hydrogenase catalysis. Since C298 is strongly conserved among all the known functional [FeFe]-hydrogenases, it is reasonable that the results obtained here for CaHydA have broader implications regarding its functional significance in the entire class. Reference: Morra S, Giraudo A, Di Nardo G, King PW, Gilardi G and Valetti F – Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase – PLoS ONE, 2012, 7(10), e48400.

Studies of Cysteine 298 Role in [FeFe]-hydrogenases

MORRA, SIMONE;VALETTI, Francesca;DI NARDO, Giovanna;GILARDI, Gianfranco
2013

Abstract

Cysteine 298 is one of the closest aminoacids to the Fed atom of the H-cluster of [FeFe]-hydrogenases. C298 was targeted in CaHydA by site saturation mutagenesis (SSM), a technique that randomly mutates the selected position into all the 20 aminoacids. The functional effects were simultaneously analysed with a specifically developed activity screening protocol, necessary to obtain quickly functional information from a large number of [FeFe]-hydrogenase variants. The screening protocol is a semi-quantitative colorimetric method that can be applied directly on E. coli colonies. Screening of the SSM library showed that the only amino acid that can replace cysteine with a remarkable activity is aspartic acid. Characterization of the purified C298D variant in comparison to the wild type revealed that the kcat decreases by 50% only and that there is no major structural effect. Interestingly, C298D causes a significant shift towards more acidic values of the pH activity profile. Characterization of the purified variants C298E, C298S, C298N and C298A, chosen because of structural similarity, further demonstrated that replacement of cysteine with other residues severely impairs the activity by 4 to 5 orders of magnitude or completely abolish it. The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and the shift in the pH activity profile demonstrate experimentally the central role of C298 in the proton transfer pathway to the active site during [FeFe]-hydrogenase catalysis. Since C298 is strongly conserved among all the known functional [FeFe]-hydrogenases, it is reasonable that the results obtained here for CaHydA have broader implications regarding its functional significance in the entire class. Reference: Morra S, Giraudo A, Di Nardo G, King PW, Gilardi G and Valetti F – Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase – PLoS ONE, 2012, 7(10), e48400.
10th International Hydrogenase Conference
Szeged (Hungary)
8-12/7/2013
10th International Hydrogenase Conference
University of Szeged
add. 48
add. 48
hydrogenase; site-directed mutagenesis; proton transfer pathway; enzyme catalysis
Morra S; Valetti F; Di Nardo G; King PW; Gilardi G
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/150072
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