The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state.
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). FS0 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.
Rothery
Richard A
RA
CIHR Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 474 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
Bertero
Michela G
MG
Cammack
Richard
R
Palak
Monica
M
Blasco
Francis
F
Strynadka
Natalie C J
NC
Weiner
Joel H
JH
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000737:chemistry; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry*; D010084:Oxidation-Reduction; D011199:Potentiometry; D021122:Protein Subunits / Q000737:chemistry*; D011622:Pterins / Q000737:chemistry
D001059:Apoproteins; D003067:Coenzymes; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D011622:Pterins; C063761:molybdopterin guanine dinucleotide; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase