title: Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.
abstract: [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Kuchenreuther
authors.forename: Simon J
authors.initials: SJ
authors.lastname: George
authors.forename: Celestine S
authors.initials: CS
authors.lastname: Grady-Smith
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Cramer
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism*; D003486:Cyanides / Q000737:chemistry / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; D007506:Iron-Sulfur Proteins; D008024:Ligands; D014443:Tyrosine; D002248:Carbon Monoxide; D006859:Hydrogen; D006864:Hydrogenase