Characterization of redox- and gas -responsive heme -thiolate proteins

• Main Author: Marvin, Katherine A
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2008
• Subjects: Biochemistry; Chemistry; Inorganic chemistry
• ISBN: 0549634649; 9780549634645

Coordination of a cysteine(thiolate) axial ligand to heme b (iron protoporphrin IX) opposite another neutral protein ligand confers a unique sensitivity to changes in the redox environment. Heme-thiolate proteins are often also functionally regulated by the binding of the gas ligands CO and/or NO, but not O2. In this work, the spectroscopic characterization of three proteins, BxRcoM-2, DmE75 and HsRev-erbβ, of this class of low-spin, 6-coordinate heme-thiolates is presented. A fourth protein, cystathionine β-synthase, which incorporates a heme-thiolate moiety and an additional cofactor at the active site for enzymatic activity, has been previously characterized in our lab, and the heme redox behavior is further investigated in this work. The aerobic bacterium Burkholderia xenovorans expresses two homologous heme-PAS proteins that demonstrate CO-dependent DNA binding in vivo. Studies of one regulator of CO metabolism, BxRcoM-2, reveals a 6-coordinate Fe(III) heme-thiolate center, which is anchored by a histidine; reduction results in replacement of the cysteine(thiolate) by another neutral ligand, likely a nearby methionine residue, that is subsequently displaced when CO or NO binds. The nuclear receptors (NRs) Drosophila melanogaster E75 and the human homolog Rev-erbβ are transcriptional silencers that regulate development, metabolism and circadian processes. In the NR superfamily, this group is unique in binding heme b. Characterization of the heme ligand environments in both NRs reveals coordination reminiscent of known heme-thiolate redox and gas sensors which replace cysteine(thiolate) with histidine when reduced. Both E75 and Rev-erbβ bind CO trans to histidine; however, E75 and Rev-erbβ form 5-and 6-coordinate NO adducts, repectively. Functional studies implicate the E75 heme in both redox-, CO- and NO-sensing, while the heme function in Rev-erbβ remains ambiguous. Human cystathionine β-synthase (hCBS) is a pyridoxal 5'-phosphate-dependent enzyme. Human CBS is unusual among PLP enzymes in binding a heme-thiolate moiety; however, heme function in this enzyme has yet to be fully elucidated. Previous work has demonstrated that the 6-coordinate heme-thiolate complex exhibits pH- and temperature-dependent behavior when reduced; perturbations of the heme ligation negatively impact hCBS activity. The studies herein investigate the influence of substrate and allosteric effector binding on processes occurring at the reduced heme.