The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state

TY - JOUR

T1 - The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state

AU - Rothery, R A

AU - Bertero, M G

AU - Cammack, R

AU - Palak, M

AU - Blasco, F

AU - Strynadka, N C J

AU - Weiner, J H

PY - 2004/5/11

Y1 - 2004/5/11

N2 - We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A(NarGHI) catalytic subunit (NarG). FSO is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures ( <15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = 3/2 ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = 1/2 and DeltaS = 3/2 transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.

AB - We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A(NarGHI) catalytic subunit (NarG). FSO is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures ( <15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = 3/2 ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = 1/2 and DeltaS = 3/2 transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.

UR - http://www.scopus.com/inward/record.url?scp=2442546646&partnerID=8YFLogxK

U2 - 10.1021/bi049938l

DO - 10.1021/bi049938l

M3 - Article

SN - 1520-4995

VL - 43

SP - 5324

EP - 5333

JO - Biochemistry

JF - Biochemistry

IS - 18

ER -