Abstract
Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN(-)) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN(-) ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)(2)(CO) entity of [NiFe] hydrogenase.
Highlights
The active site iron of [NiFe] hydrogenases is equipped with a carbonyl ligand (CO) and two cyanides (CNϪ)
We show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the carbon monoxide (CO) ligand
The results reveal that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)2(CO) entity of the active site in [NiFe] hydrogenases and provide insights into the chronological order of ligand binding to the HypCD complex
Summary
The active site iron of [NiFe] hydrogenases is equipped with a carbonyl ligand (CO) and two cyanides (CNϪ). Three unusual diatomic ligands in the form of two cyanides (CN؊) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. The MBH of R. eutropha represents an exception as the Fe(CN)2CO moiety is first transferred to the MBH-specific scaffold protein HoxV and subsequently to the apo-form of the large subunit (Fig. 1A) [11]. The overall process of the [NiFe]-active site assembly is fairly established, major questions concerning the origin of carbon monoxide, the sequence of events, and the precise function of the central HypCD complex remained open.
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