sup 1 H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b sub 5

Journal Article · Mon Feb 18 23:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00221a021· OSTI ID:5510256

Lee, Kangbong; La Mar, G N; Pandey, R K; Rezzano, I N; Mansfield, K E; Smith, K M ^[1]; Pochapsky, T C; Sligar, S G ^[2]

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1. Univ. of California, Davis (USA)
2. Univ. of Illinois, Urbana (USA)

{sup 1}H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b{sub 5} reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl){sub 8-n}porphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b{sub 5} is judged much less polar and less sterically accommodating than that of myoglobin. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.

• OSTI ID: 5510256
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:7; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BARYONS
CARBOXYLIC ACIDS
CYTOCHROMES
ELEMENTARY PARTICLES
FERMIONS
GLOBINS
HADRONS
HEAVY WATER
HEME
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYPERFINE STRUCTURE
MAGNETIC RESONANCE
MAMMALS
MYOGLOBIN
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OVERHAUSER EFFECT
OXYGEN COMPOUNDS
PIGMENTS
PORPHYRINS
PROTEINS
PROTONS
RATS
RESONANCE
RODENTS
VERTEBRATES
WATER