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^,

Abstract

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.

Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

“…The unconstrained geometry of cytochrome b 5 -like hemichromes is also inferred from the values of the Ne 2prox -Fe-Ne 2dist angle, which measures the deviation from linearity of the apical bonds at the iron atom. These values are very close to 1808, if we do not consider the case of Rattus norvegicus cyt b 5 (Ne 2prox -FeNe 2dist 5 1668), whose structure has been determined at a resolution significantly lower than the other members of the set (37). Deviations from the ideal His coordination are generally pronounced in Hbs, and more so in tetrameric than monomeric Hbs.…”

A further investigation of the cytochrome b 5–cytochrome c complex

“…Some rearrangements could take place in solution, e.g. bringing the side chain of Lys72 of cytochrome c close to heme propionate 6 of cytochrome b 5 , as predicted by calculations [7] and also suggested by experiments on cytochrome b 5 [67]. On the other hand, residues 27 and 79 of cytochrome c are too far from the interface in our calculated model to account for their role, proposed on the basis of experimental data, in modulating electron transfer between cytochrome b 5 and cytochrome c. A possible explanation for this finding is that the complex proposed by Salemme, which is competent for electron transfer, forms dynamically in solution, but only exists for a fraction of time small enough not to give rise to appreciable perturbation of NMR chemical shifts and of 15 N relaxation rates.…”

The Crystal Structure of Synechocystis Hemoglobin with a Covalent Heme Linkage

“…6 shows the azimuthal angles for the above hxHbs and the two SynHb structures. Myoglobin (Mb) (49) and leghemoglobin (Lba) (50) are also shown, along with four cytochrome b 5 proteins; bovine (bCYT) and rat (rCYT) cytochrome b 5 (51,52), and the cytochrome b 5 domains from human (HSO) and chicken liver (CSO) sulfite oxidases (53,54).…”