Myoglobin and cytochrome b5: A nuclear magnetic resonance study of a highly dynamic protein complex

Jonathan A.R. Worrall, Yijeng Liu, Peter B. Crowley, Judith M. Nocek, Brian M Hoffman, Marcellus Ubbink*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

Abstract

The transient complex of bovine myoglobin and cytochrome b5 has been investigated using a combination of NMR chemical shift mapping, 15N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b5 amide resonances upon complex formation with either metmyoglobin (FeIII) or carbon monoxide-bound myoglobin (FeII) are more than 10-fold smaller than in other transient redox protein complexes. From 15N relaxation experiments, an increase in the overall correlation time of cytochrome b5 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 b5 titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a Ka in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b5, 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.

Original languageEnglish (US)
Pages (from-to)11721-11730
Number of pages10
JournalBiochemistry
Volume41
Issue number39
DOIs
StatePublished - Oct 1 2002

ASJC Scopus subject areas

  • Biochemistry

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