The complex of cytochrome f and plastocyanin from Nostoc sp. PCC 7119 is highly dynamic.
Bottom Line: The complex from Nostoc sp.Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc.The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.
Affiliation: Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.Show MeSH
Related in: MedlinePlus
Mentions: 15N-enriched Pc was produced in a cytoplasmic expression system (seethe Experimental Section) with a ZnII ion in the copper binding siteto eliminate the paramagnetic effect of CuII and possibleinterference from electron transfer reactions. To establish whether the introduction of a probe interfereswith the Pc–Cyt f interaction, chemical shift analyseswere carried out for all variants. First, Zn-substituted [15N]Pc wastitrated with wild-type Cyt f, and HSQC spectra were acquiredat each titration point. The binding constant was obtained by fitting thechemical shift perturbation curves for the most affected amide groups (Figure 2 A), thereby yielding aKd of 8(3)×10−5m (estimated experimental error in parentheses), similar to thereported values of 3.8(0.1)×10−5m for Cu-Pc and6.2(0.9)×10−5m for Cd-substituted Pc.
Affiliation: Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.