TY  - JOUR
A1  - Schrapers, Peer
A1  - Hartmann, Tobias
A1  - Kositzki, Ramona
A1  - Dau, Holger
A1  - Reschke, Stefan
A1  - Schulzke, Carola
A1  - Leimkühler, Silke
A1  - Haumann, Michael
T1  - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus
JF  - Inorganic chemistry
N2  - Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
Y1  - 2015
U6  - https://doi.org/10.1021/ic502880y
SN  - 0020-1669
SN  - 1520-510X
VL  - 54
IS  - 7
SP  - 3260
EP  - 3271
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - JOUR
A1  - Hahn, Aaron
A1  - Engelhard, Christopher
A1  - Reschke, Stefan
A1  - Teutloff, Christian
A1  - Bittl, Robert
A1  - Leimkühler, Silke
A1  - Risse, Thomas
T1  - Structural Insights into the Incorporation of the Mo Cofactor into Sulfite Oxidase from Site-Directed Spin Labeling
JF  - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition
N2  - Mononuclear molybdoenzymes catalyze a broad range of redox reactions and are highly conserved in all kingdoms of life. This study addresses the question of how the Mo cofactor (Moco) is incorporated into the apo form of human sulfite oxidase (hSO) by using site-directed spin labeling to determine intramolecular distances in the nanometer range. Comparative measurements of the holo and apo forms of hSO enabled the localization of the corresponding structural changes, which are localized to a short loop (residues 263-273) of the Moco-containing domain. A flap-like movement of the loop provides access to the Moco binding-pocket in the apo form of the protein and explains the earlier studies on the in vitro reconstitution of apo-hSO with Moco. Remarkably, the loop motif can be found in a variety of structurally similar molybdoenzymes among various organisms, thus suggesting a common mechanism of Moco incorporation.
KW  - biocatalysis
KW  - cofactors
KW  - enzymes
KW  - EPR spectroscopy
KW  - protein structures
Y1  - 2015
U6  - https://doi.org/10.1002/anie.201504772
SN  - 1433-7851
SN  - 1521-3773
VL  - 54
IS  - 40
SP  - 11865
EP  - 11869
PB  - Wiley-VCH
CY  - Weinheim
ER  -