¹⁵N chemical shift changes in cytochrome b₅: redox-dependent vs. guanidinium chloride-induced changes

Abstract.

The origin of the recently reported chemical shift changes of backbone amide nitrogens of redox proteins upon redox state changes has been investigated. These effects are particularly marked in cytochromes and are clearly present after correction for pseudocontact shifts in the oxidized form (Boyd J, Dobson CM, Morar AS, Williams RJP, Pielak GJ (1999) J Am Chem Soc 121:9247–9248; Guiles RD, Basus VJ, Sarma S, Malpure S, Fox KM, Kuntz ID, Waskell L (1993) Biochemistry 32:8329–8340). ¹⁵N-HSQC experiments have been performed on both oxidized and reduced forms of cytochrome b₅ in the absence and in the presence of 2 M guanidinium chloride (GdmCl). GdmCl in this concentration is known to sizably alter the structure of the oxidized form of the protein and, in particular, to perturb the hydrogen bonding network. However, the perturbation of the ¹⁵N-NMR chemical shift changes is minor compared to the changes occurring upon reduction. It is concluded that changes in hydrogen bonding upon reduction must be modest and cannot account for the observed chemical shift effects. Alternative explanations should thus be looked for.