Die pleiotrope Maturation der sauerstofftoleranten [NiFe]-Hydrogenasen aus Ralstonia eutropha

Hydrogenasen sind komplexe Enzyme, die die reversible Oxidation von molekularem Wasserstoff zu Protonen und Elektronen katalysieren. Diese Enzyme erlauben ihrem Wirtsorganismus das Wachstum unter chemolithoautotrophen Bedingungen. Der Modellorganismus Ralstonia eutropha besitzt drei gut charakterisierte Hydrogenasen der [NiFe]-Klasse, die sich durch ihre Sauerstofftoleranz auszeichnen. Ihr aktives Zentrum besteht aus einer komplexen prosthetischen Gruppe, welche aus einem Nickel- und einem Eisenatom besteht. Letzteres koordiniert drei diatomare Liganden, zwei Cyanide und ein CO. Die Synthese der gesamten Ni(SR)2(µ-SR)2Fe(CN)2(CO)-Gruppe ist ein komplexer Prozess. Die sogenannte Maturation benötigt wenigstens sechs akzessorische Proteine, die sogenannten Hyp-Proteine. Das umfassende Verständnis dieser Maturationsprozesse ermöglicht eine Vielzahl von biotechnologischen Anwendungen. Die vorliegende Arbeit untersucht die Maturation unter verschiedenen Gesichtspunkten. Zentrale, offene Fragen sind die Herkunft des Carbonylliganden sowie die Prozesse, die zur Ligandierung des katalytischen Eisens führen. Dazu wurden molekularbiologische, biochemische und spektroskopische Methoden in Verbindung mit Isotopenmarkierung eingesetzt. Unter anderem konnte dabei gezeigt werden, dass das katalytische Eisen alle seine Liganden bereits im HypCD-Komplex, dem zentralen Element der Maturation, erhält. Ferner konnte in dieser Arbeit, erstmalig für [NiFe]-Hydrogenasen, eine konkrete Biosynthese des seltenen und toxischen diatomaren CO-Liganden beschrieben werden. Ausgehend vom Alpha-Kohlenstoff von Glycin wird der Tetrahydrofolat (THF)-abhängige C1-Metabolismus mit C1-Einheiten versorgt. Durch die enzymatische Aktivität von HypX wird die Formylgruppe von N10-Formyl-THF zu CO umgesetzt.Hydrogenases are complex enzymes that catalyze the reversible oxidation of molecular hydrogen into protons and electrons. These enzymes allow their host organism to grow under chemolithoautotrophic conditions. The model organism Ralstonia eutropha has three well-characterized [NiFe]-hydrogenases, which exhibit an extraordinary high oxygen tolerance. Its active center is a complex prosthetic group which consists of a nickel and iron atom. The latter coordinates three diatomic ligands, two cyanides and one CO. The biosynthesis of the whole Ni(SR)2(μ-SR)2Fe(CN)2(CO)-group is a complex process. This so-called maturation process needs the activity of at least six accessory proteins, the Hyp-proteins. Understanding the maturation allows a variety of biotechnological applications. The present study examines the maturation of [NiFe]-hydrogenases under different aspects. The major questions concern the origin of the carbonyl ligand as well as the processes that lead to ligandation of the designated catalytic iron. To adress these tasks, molecular biological, biochemical and spectroscopic methods in combination with isotopic labeling were employed. Inter alia, it could be shown that the catalytic iron in the HypCD-complex, the central element of the maturation process, contains all three diatomic ligands. Furthermore, this study describes, for the first time in [NiFe]-hydrogenases, a specific biosynthetic route of the rare and toxic diatomic CO-ligand. Starting from the alpha-carbon of glycine the tetrahydrofolate (THF)-dependent one-carbon metabolism is replenished with one-carbon units. Subsequently the formyl group from N10-formyl-THF is hydrolyzed by the enzymatic activity from HypX and further converted to carbon monoxide as determined by isotopic labeling and infrared spectroscopy

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster

Hydrogenases catalyze the redox interconversion of protons and H(2), an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S](H) subcluster coupled to a [2Fe](H) subcluster bound by CO, CN(–), and azadithiolate ligands. The [2Fe](H) subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)(2)(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe–4S](aux)[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)(2)(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release