WU, JZ LAMAR, GN YU, LP LEE, KB WALKER, FA CHIU, ML SLIGAR, SG 2024-01-21T23:46:53Z 2024-01-21T23:46:53Z 2022-01-10 1991-02-26 0006-2960 https://pubs.kist.re.kr/handle/201004/146834 The solution 500-MHz H-1 NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25-degrees-C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved H-1 NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25-degrees-C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin reversible high spin equilibrium with an intact His-Fe-Met coordination . The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations. English AMER CHEMICAL SOC NUCLEAR MAGNETIC-RESONANCE INDIVIDUAL H-1-NMR ASSIGNMENTS C-TYPE CYTOCHROMES PROTON NMR COORDINATION GEOMETRY HEME IRON PSEUDOMONAS-MENDOCINA CONFORMATION CHANGE AXIAL METHIONINE AMINO-ACIDS H-1-NMR STUDY OF THE SOLUTION MOLECULAR AND ELECTRONIC-STRUCTURE OF ESCHERICHIA-COLI FERRICYTOCHROME-B562 - EVIDENCE FOR S = 1/2 REVERSIBLE S = 5/2 SPIN EQUILIBRIUM FOR INTACT HIS MET LIGATION Article 10.1021/bi00222a020 1 BIOCHEMISTRY, v.30, no.8, pp.2156 - 2165 BIOCHEMISTRY 30 8 2156 2165 scie scopus A1991EZ60200020 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article NUCLEAR MAGNETIC-RESONANCE INDIVIDUAL H-1-NMR ASSIGNMENTS C-TYPE CYTOCHROMES PROTON NMR COORDINATION GEOMETRY HEME IRON PSEUDOMONAS-MENDOCINA CONFORMATION CHANGE AXIAL METHIONINE AMINO-ACIDS