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PDBsum entry 1b5m
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Electron transport
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PDB id
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1b5m
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References listed in PDB file
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Key reference
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Title
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13c nmr spectroscopic and X-Ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.
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Authors
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M.J.Rodríguez-Marañón,
F.Qiu,
R.E.Stark,
S.P.White,
X.Zhang,
S.I.Foundling,
V.Rodríguez,
C.L.Schilling,
R.A.Bunce,
M.Rivera.
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Ref.
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Biochemistry, 1996,
35,
16378-16390.
[DOI no: ]
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PubMed id
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Abstract
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The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme
propionate groups in the electrostatic binding between OM cytochrome b5 and
horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray
crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was
expressed in Escherichia coli as previously described [Rivera M., Walker, F.A.
(1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from
the heme propionate carbons in ferricytochrome b5 was carried out by a
combination of one- and two-dimensional NMR experiments. Titrations of
[13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried
out in order to monitor the resonances arising from the heme propionate carbonyl
carbons in OM cytochrome b5. The results from these titrations clearly show that
only the heme propionate located on the exposed heme edge in OM cytochrome b5
participates in the electrostatic stabilization of the complex between OM
cytochrome b5 and horse heart cytochrome c. Similar experiments carried out
monitoring 13C resonances arising from several other heme substituents
demonstrated that the stoichiometry of the complex is 1:1. A conditional binding
constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was
obtained for the formation of the complex by fitting the binding curves obtained
experimentally to a model based on this stoichiometry. The X-ray crystal
structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that
the structures of bovine liver microsomal cytochrome b5 and rat liver OM
cytochrome b5 are almost identical when compared at medium resolution. The
similarity between the two structures, combined with the findings that only the
heme propionate located on the exposed heme edge of OM cytochrome b5
participates in the electrostatic binding to cytochrome c and that the stability
of this complex is similar to that measured for the association between
microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of
interaction on OM cytochrome b5 is almost identical to the one elucidated for
microsomal cytochrome b5. It is therefore possible to conclude that the large
body of information gathered by many investigators for the nonphysiological
interaction between microsomal cytochrome b5 and cytochrome c (recently
reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995)
Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical
validity.
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