Abstract

The initial electron transfer time in the photosynthetic reaction centre of Rhodobacter sphaeroides is highly sensitive to the replacement of tyrosine M210 by a tryptophan residue. Low-temperature magic angle spinning 13C NMR is used to study Rhodobacter sphaeroides 2.4.1 (M)Y210W mutant reaction centres that are labelled with [4′- 13C]tyrosine. The response of (M)Y210 in R26 is assigned unambiguously to the most upfield narrow signal (linewidth 34 Hz) at σ i = 152.2 ppm, in the region where non-hydrogen bonded tyrosine signals are expected. From the comparison with the signal of labelled R26 it follows that the chemical environment of the (M)Y210 label is unique. The Y(M)210 is in a structurally and electrostatically homogeneous region on the sensitivity scale of the MAS NMR technique. The environment of M210 is structurally stable and the observation of a narrow line shows that the (M)Y210 side chain can be considered static with respect to rotational diffusion on time scales as long as 10 −2s. The narrow signals from the remaining labels in the protein interior for the 2.4.1 (M)Y210W mutant are remarkably similar to those observed for R26. Using a commonly accepted ratio of 150 ppm shift per positive charge equivalent for aromatic carbons, the chemical shift differences between mutant and R26 translate into small variations of the order of 10 −3 electronic equivalents of charge polarization. Also the linewidths are similar, except for a narrow response at σ i = 156.4 ppm that sharpens slightly in the (M)Y210W mutant. Thus, the influence of the (M)Y210W mutation on the global electrostatic properties and structure of the protein, as probed by the tyrosine labels, is minimal. This strongly argues against an explanation of slow and non-exponential electron transfer kinetics in the (M)Y210W RC in terms of loss of structural integrity upon mutation. In contrast, the NMR results strongly support current opinions that (M)Y210 contributes to the fine-tuning of the energy levels of prosthetic groups involved in electron transfer.

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