The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO)2(CN)] organometallic precursor

The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe–Fe binuclear subcluster: CN−, CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren’t fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO)2(CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO)2(CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13CN-containing carrier affords 13CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a 13C3,15N-Cys–labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1–Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.

MOLECULAR DYNAMICS STUDY OF THE TRANSCRIPTIONAL RECOGNITION MECHANISM OF HEME ACTIVATE PROTEIN (HAP)

The heme activate protein (HAP) is a model system for understanding protein–DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of HAP, the specific recognition mechanism of the target DNA by HAP has remained elusive. This paper gives the results of a study using molecular dynamics simulations performed for a single DNA fragment (USACYC7) and three protein–DNACYC7complex crystal structures: the HAP-wt and two HAP mutants — HAP-PC7: S63G; HAP-18: S63R. Comparative molecular dynamics simulations reveal that the distributions of protein–DNA interactions recognizing the key base steps (CGC) are consistent with their transcriptional activities. Relative to the similar conformations of three bound DNA, the different flexibilities in involving DNA recognition regions: N-term Arm and Zn 2 Cys 6 Binuclear Cluster in three HAPs may result in a variety of protein–DNA recognitions. Despite different intensities of motions, the essential dynamics (ED) analysis shows that the internal motions of three protein–DNA complexes are similar: three proteins all slide along DNA to find their target sites. Thus, under this condition, during the recognition process, the flexibility of the DNA recognizing regions (N-term Arm and Zn 2 Cys 6 Binuclear Cluster regions) plays a crucial role in determining the abilities of protein's recognizing DNA: the higher is its flexibility, the faster it slides along the DNA to find the targeted DNA.