C-13 MAS NMR EVIDENCE FOR A HOMOGENEOUSLY ORDERED ENVIRONMENT OF TYROSINE-M210 IN REACTION CENTERS OF RHODOBACTER-SPHAEROIDES

The initial electron transfer time in the photosynthetic reaction cent re of Rhodobacter sphaeroides is highly sensitive to the replacement o f tyrosine M210 by a tryptophan residue. Low-temperature magic angle s pinning C-13 NMR is used to study Rhodobacter sphaeroides 2.4.1 (M)Y21 0W mutant reaction centres that are labelled with [4'-C-13]tyrosine. T he response of (M)Y210 in R26 is assigned unambiguously to the most up field narrow signal (linewidth 34 Hz) at sigma(i) = 152.2 ppm, in the region where non-hydrogen bonded tyrosine signals are expected. From t he comparison with the signal of labelled R26 it follows that the chem ical environment of the (M)Y210 label is unique. The Y(M)210 is in a s tructurally and electrostatically homogeneous region on the sensitivit y scale of the MAS NMR technique. The environment of M210 is structura lly stable and the observation of a narrow line shows that the (M)Y210 side chain can be considered static with respect to rotational diffus ion on time scales as long as 10(-2) s. The narrow signals from the re maining labels in the protein interior for the 2.4.1 (M)Y210W mutant a re remarkably similar to those observed for R26. Using a commonly acce pted ratio of 150 ppm shift per positive charge equivalent for aromati c carbons, the chemical shift differences between mutant and R26 trans late into small variations of the order of 10(-3) electronic equivalen ts of charge polarization. Also the linewidths are similar, except for a narrow response at sigma(i) = 156.4 ppm that sharpens slightly in t he (M)Y210W mutant. Thus, the influence of the (M)Y210W mutation on th e global electrostatic properties and structure of the protein, as pro bed by the tyrosine labels, is minimal. This strongly argues against a n explanation of slow and non-exponential electron transfer kinetics i n the (M)Y210W RC in terms of loss of structural integrity upon mutati on. In contrast, the NMR results strongly support current opinions tha t (M)Y210 contributes to the fine-tuning of the energy levels of prost hetic groups involved in electron transfer.