TY - JOUR
T1 - The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO)2(CN)] organometallic precursor
AU - Rao, Guodong
AU - Pattenaude, Scott A.
AU - Alwan, Katherine
AU - Blackburn, Ninian J.
AU - Britt, R. David
AU - Rauchfuss, Thomas B.
N1 - Funding Information:
ACKNOWLEDGMENTS. This work was supported by NIH Grants 1R35GM126961-01 (to R.D.B.), GM61153 (to T.B.R.), and R01GM123725 (to N.J.B.). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences Contract DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41 GM103393). We thank Prof. Frank E. Osterloh and Dr. Zeqiong Zhao (University of California, Davis) for the help with GC detection of H2; and Dr. Lucas Li and Dr. Alexander Ulanov at the Roy J. Carver Biotechnology Center (University of Illinois Urbana– Champaign) for help with pyruvate detection and quantification.
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/10/15
Y1 - 2019/10/15
N2 - 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.
AB - 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.
KW - Cysteine
KW - H-cluster biosynthesis
KW - Iron carbonyl cyanides
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U2 - 10.1073/pnas.1913324116
DO - 10.1073/pnas.1913324116
M3 - Article
C2 - 31570604
AN - SCOPUS:85073302696
SN - 0027-8424
VL - 116
SP - 20850
EP - 20855
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 42
ER -