Myoglobin and Cytochrome b₅:  A Nuclear Magnetic Resonance Study of a Highly Dynamic Protein Complex

Abstract

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b(5) titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a K(a) in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b(5), with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.

The Interactions of Cyanobacterial Cytochromec6 and Cytochrome f, Characterized by NMR

“…Line broadening effects on the order of 2 Hz were observed (Fig. 3B), consistent with a weak and highly dynamic interaction (36). Increasing the ionic strength by the addition of 200 mM NaCl had no effect on the protein interactions.…”

Structure of the Complex between Plastocyanin and Cytochrome f from the Cyanobacterium Nostoc sp. PCC 7119 as Determined by Paramagnetic NMR

“…The overall sizes of the perturbations and the localized nature of the binding map indicate that the complex between PCd and Cf is well defined according to the classification for well defined versus dynamic complexes, suggested by Worrall et al (55) and Prudêncio and Ubbink (56). That is, PCd adopts a single predominant conformation during most of the lifetime of the complex.…”

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

“…In this respect, it is also worth noting that the interface regions on cytochrome c in the Salemme model and in the electrostatically dominant model are adjacent. The importance of inter-protein dynamics in electron transfer complexes appears to be quite different in different systems [14,24,25], but likely plays a crucial role in modulating the rate of the process [68].…”

“…In a similar approach, heteronuclear NMR and docking calculations were used for building structural models of the cytochrome c 553 -ferredoxin complex and a combination of TROSY experiments and docking calculations were used for the study of the [Fe]-hydrogenase-cytochrome c 553 complex [22,23]. Other, more recent, applications of similar strategies include the cytochrome b 5 -myoglobin [24] and the cytochrome ccytochrome f [25] complexes. The present approach can thus be seen as a general strategy for the investigation of adducts between electron transfer proteins.…”