Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex.

Clarke, Thomas A. ORCID: https://orcid.org/0000-0002-6234-1914, Dennison, Victoria, Seward, Harriet E., Burlat, Bénédicte, Cole, Jeffrey A., Hemmings, Andrew M. ORCID: https://orcid.org/0000-0003-3053-3134 and Richardson, David J. ORCID: https://orcid.org/0000-0002-6847-1832 (2004) Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex. Journal of Biological Chemistry, 279 (40). pp. 41333-41339. ISSN 0021-9258

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Abstract

Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically. This study reports the first purification of NrfB, a novel 20-kDa pentaheme c-type cytochrome encoded by the nrfB gene that follows the nrfA gene in many bacterial nrf operons. Analyses by gel filtration demonstrated that NrfB purifies as a decaheme homodimer. Analysis of NrfB by UV-visible and magnetic circular dichroism spectroscopy demonstrates that all five NrfB ferric heme irons are low spin and are most likely coordinated by two axial histidine ligands. Spectropotentiometry revealed that the midpoint redox potentials of five ferric hemes were in the low potential range of 0 to –400 mV. Analysis by low temperature EPR spectroscopy revealed signals that arise from two classes of bis-His ligated low spin hemes, namely a rhombic trio at g1,2,3 = 2.99, 2.27, and 1.5 that arises from two hemes in which the planes of histidine imidazole rings are near-parallel and a large gmax signal at g = 3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular. NrfB was also overexpressed as a recombinant protein, which had similar spectropotentiometric properties as the native protein. Reconstitution experiments demonstrated that the reduced decaheme NrfB dimer could serve as a direct electron donor to the oxidized decaheme NrfA dimer, thus forming a transient 20-heme [NrfB]2[NrfA]2 electron transfer complex.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry (former - to 2024)
UEA Research Groups:	Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Organisms and the Environment Faculty of Science > Research Groups > Plant Sciences Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Energy Materials Laboratory
Depositing User:	EPrints Services
Date Deposited:	01 Oct 2010 13:37
Last Modified:	24 Sep 2024 10:01
URI:	https://ueaeprints.uea.ac.uk/id/eprint/926
DOI:	10.1074/jbc.M407604200

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