Adenosine triphosphate-induced electron transfer in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.
2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans catalyzes the chemical difficult elimination of water from (R)-2-hydroxyglutaryl-CoA to glutaconyl-CoA. The enzyme consists of two oxygen-sensitive protein components, the homodimeric activator (A) with one [4Fe-4S]1+/2+ cluster and the heterodimeric dehydratase (D) with one nonreducible [4Fe-4S]2+ cluster and reduced riboflavin 5'-monophosphate (FMNH2). For activation, ATP, Mg2+, and a reduced flavodoxin (16 kDa) purified from A. fermentans are required. The [4Fe-4S](1+/2+) cluster of component A is exposed to the solvent since it is accessible to iron chelators. Upon exchange of the bound ADP by ATP, the chelation rate is 8-fold enhanced, indicating a large conformational change. Oxidized component A exhibits ATPase activity of 6 s(-1), which is completely abolished upon reduction by one electron. UV-visible spectroscopy revealed a spontaneous one-electron transfer from flavodoxin hydroquinone (E(0)' = -430 mV) to oxidized component A, whereby the [4Fe-4S]2+ cluster of component A became reduced. Combined kinetic, EPR, and Mössbauer spectrocopic investigations exhibited an ATP-dependent oxidation of component A by component D. Whereas the [4Fe-4S]2+ cluster of component D remained in the oxidized state, a new EPR signal became visible attributed to a d1-metal species, probably Mo(V). Metal analysis with neutron activation and atomic absorption spectroscopy gave 0.07-0.2 Mo per component D. In summary, the data suggest that in the presence of ATP one electron is transferred from flavodoxin hydroquinone via the [4Fe-4S]1+/2+ cluster of component A to Mo(VI) of component D, which is thereby reduced to Mo(V). The latter may supply the electron necessary for transient charge reversal in the unusual dehydration.
Hans
Marcus
M
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
Bill
Eckhard
E
Cirpus
Irina
I
Pierik
Antonio J
AJ
Hetzel
Marc
M
Alber
Dorothea
D
Buckel
Wolfgang
W
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D005418:Flavodoxin / Q000737:chemistry; D006836:Hydro-Lyases / Q000378:metabolism*; D006868:Hydrolysis; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D005418:Flavodoxin; D000255:Adenosine Triphosphate; C054464:2-hydroxyglutaryl-CoA dehydratase; D006836:Hydro-Lyases