title: A one-residue switch reverses the orientation of a heme b cofactor. Investigations of the ferriheme NO transporters nitrophorin 2 and 7 from the blood-feeding insect Rhodnius prolixus.
abstract: This study represents the identification of a single amino acid residue that has the major responsibility for the isomeric orientation of a heme b cofactor in a ferriheme protein. The insertion of hemin b into the asymmetric environment of a protein pocket facilitates two cofactor orientations, A and B, which is often called "heme rotational disorder". The proteins studied herein are nitrophorins, a class of ferriheme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus, in this case NP2 and NP7. NMR spectroscopy (pH* 5.5) of the imidazole complex of NP7 revealed solely the A orientation, whereas NP2 shows primarily the B orientation ( approximately 1:5 A:B). The glutamate 27 residue in NP7 is an obvious difference in the heme pocket compared to those of NP1-4, all of which present a valine residue [valine 24 (NP2 and NP3) or valine 25 (NP1 and NP4)] at the same position. Consequently, the mutant NP2(V24E) was prepared and shown to reverse the heme orientation to exclusively A, whereas NP7(E27V) revealed an approximately 1:3 A:B ratio. The reversal A <--> B following the change glutamine <--> valine was further indicated in circular dichroism (CD) spectroscopy with a positive (A) or negative (B) Deltaepsilon of the heme Soret band. Moreover, CD spectroscopy was applied to the mutant NP7(E27Q) and indicated mainly the A orientation, which allows us to conclude that the steric hindrance provided by the glutamate residue is responsible for the heme orientation rather then the carboxylate charge.
authors.affiliation: Department of Chemistry, The University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721-0041, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Yang
authors.forename: Hongjun
authors.initials: H
authors.lastname: Zhang
authors.forename: Markus
authors.initials: M
authors.lastname: Knipp
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006427:Hemin / Q000737:chemistry* / Q000502:physiology; D019476:Insect Proteins / Q000737:chemistry* / Q000502:physiology; D026901:Membrane Transport Proteins / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012239:Rhodnius / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid
chemical_list: D006420:Hemeproteins; D019476:Insect Proteins; D026901:Membrane Transport Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D006418:Heme; D006427:Hemin