The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis.
Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2production. In contrast to [NiFe] hydrogenases, they display reduced H2inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Marta C
0000-0002-5093-5918
MC
Marques
Instituto de Catálisis y Petroleoquímica (CSIC), Madrid, Spain.
Cristina
C
Tapia
Instituto de Catálisis y Petroleoquímica (CSIC), Madrid, Spain.
Oscar
O
Gutiérrez-Sanz
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Ana Raquel
AR
Ramos
Biochemistry Department, University of Missouri, Columbia, Missouri, USA.|Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA), Berkeley, California, USA.
Kimberly L
KL
Keller
Biochemistry Department, University of Missouri, Columbia, Missouri, USA.|Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA), Berkeley, California, USA.
Judy D
0000-0002-9152-5544
JD
Wall
Instituto de Catálisis y Petroleoquímica (CSIC), Madrid, Spain.
Antonio L
AL
De Lacey
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.|Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.
Pedro M
PM
Matias
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Inês A C
0000-0003-3283-4520
IAC
Pereira
D055162:Biocatalysis; D016969:Desulfovibrio vulgaris / Q000201:enzymology* / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017279:Selenocysteine / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D017279:Selenocysteine; C058560:nickel-iron-selenium hydrogenase; D006864:Hydrogenase