title: Exploiting the conformational flexibility of leghemoglobin: a framework for examination of heme protein axial ligation.
abstract: We have exploited the intrinsic conformational flexibility of leghemoglobin to reengineer the heme active site architecture of the molecule by replacement of the mobile His61 residue with tyrosine (H61Y variant). The electronic absorption spectrum of the ferric derivative of H61Y is similar to that observed for the phenolate derivative of the recombinant wild-type protein (rLb), consistent with coordination of Tyr61 to (high-spin) iron. EXAFS data clearly indicate a 6-coordinate heme geometry and a Fe-O bond length of 185pm. MCD and EPR spectroscopies are consistent with this assignment and support ligation by an anionic (tyrosinate) group. The alteration in heme ligation leads to a 148mV decrease in the reduction potential for H61Y (-127+/-5mV) compared to rLb and destabilisation of the functional oxy-derivative. The results are discussed in terms of our wider understanding of other heme proteins with His-Tyr ligation.
authors.affiliation: Department of Chemistry, University of Leicester, University Road, LE1 7RH, England, Leicester, UK.
authors.forename: Neesha
authors.initials: N
authors.lastname: Patel
authors.forename: Harriet E
authors.initials: HE
authors.lastname: Seward
authors.forename: Agneta
authors.initials: A
authors.lastname: Svensson
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Gurman
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: Emma Lloyd
authors.initials: EL
authors.lastname: Raven
mesh_terms: D001665:Binding Sites; D006420:Hemeproteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007874:Leghemoglobin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013025:Glycine max / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D006420:Hemeproteins; D007874:Leghemoglobin; D011994:Recombinant Proteins; D014443:Tyrosine; D006639:Histidine