Magneto-optical studies of RirA and cytochrome cd1

Holmes, John Derick (2012) Magneto-optical studies of RirA and cytochrome cd1. Doctoral thesis, University of East Anglia.

[img]
Preview
PDF
Download (4MB) | Preview

    Abstract

    Rhizobia are members of the Gram-negative α-proteobacteria. Rhizobium
    leguminosarum controls genes involved in iron uptake through repression under
    conditions of iron sufficiency. This part of iron regulation is co-ordinated by the
    dimeric rhizobial iron regulator, RirA. The sequence of RirA contains four cysteines
    in close proximity, three of which have been shown to be essential to the regulatory
    functions of RirA and have been proposed as the binding site of an iron-sulfur
    cluster. RirA has been overexpressed, purified and reconstituted with an ironsulphur
    cluster. EPR spectroscopy combined with iron and sulphide assays suggests
    that the protein binds a [4Fe-4S] or [3Fe-4S] cluster per monomer.
    Cytochrome cd1 is a homodimeric nitrite reductase found in denitrifying
    bacteria. Each monomer binds a heme c that transfers electrons to the structurally
    unique heme d1 at the active site. It is shown that the inactive as-prepared form of
    cd1 from Paracoccus pantotrophus is activated by pre-exposure to the substrate
    NO2
    −. Although oxidised cd1 from Pseudomonas aeruginosa does not require
    similar activation, it undergoes the same reaction with NO2
    − to produce a novel form
    of nitrosyl-heme d1. The NO derivative of Pseudomonas aeruginosa cd1 was
    prepared and characterised using variable-temperature variable-field (VTVF)
    Magnetic Circular Dichroism (MCD) spectroscopy. The novel heme d1 nitrosyl has
    thus been identified as an unprecedented FeIII-NO• species. In absorption spectra,
    this form of nitrosyl-heme d1 is indistinguishable from the low-spin FeIII state. But
    simulation of EPR spectra of the oxidised cd1s has allowed quantitation of the two
    hemes and shows that there are no populations of the FeIII-NO• product-bound active
    site heme.

    Item Type: Thesis (Doctoral)
    Faculty \ School: Faculty of Science > School of Chemical Sciences and Pharmacy
    Depositing User: Mia Reeves
    Date Deposited: 20 Dec 2012 10:24
    Last Modified: 20 Dec 2012 10:24
    URI: https://ueaeprints.uea.ac.uk/id/eprint/40457
    DOI:

    Actions (login required)

    View Item