Stark spectroscopy of the Rhodobacter sphaeroides reaction center heterodimer mutant.

Abstract

The effect of an electric field has been measured on the absorption spectrum (Stark effect) of the heterodimer mutant (M)H202L of Rhodobaeter sphaeroides reaction centers, where the primary electron donor consists of one bacteriochlorophyll a and one bacteriopheophytin a. The electronic absorption spectrum of the heterodimer mutant from 820-950 nm is relatively featureless in a poly(vinyl alcohol) film, but it exhibits some structure in a glycerol/water -ass at 77 K. A feature is seen in the Stark effect spectrum of the heterodimer at 77 K centered at 927 and 936 nm in poly(vinyl alcohol) and a glycerol/water glass, respectively. This feature has approximately the same shape and width as the Stark effect for the primary electron donor of the wild type, which consists of a pair of bacteriochlorophyll a molecules. The angle M4 between the transition moment at the frequency of absorption and the difference dipole AIA is 36± + in the wild ty and 32 ± 2 for that feature in the heterodimer. A range of values for IAAI = (13-17)/f Debye units (where f is the local field correction) is obtained for the 936-nm feature in glycerol/water, depending on analysis method. This feature is interpreted as arising from a transition to the lower exciton state of the heterodimer, which is more strongly mixed with a low-lying charge transfer transition than in the wild type.With the recent availability of genes and deletion strains for the photosynthetic bacteria Rhodobacter capsulatus (1, 2) and Rhodobacter sphaeroides (3, 4), amino acid residues in the reaction center (RC) can be manipulated by site-directed mutagenesis to investigate specific aspects of proteinprosthetic group interactions. In R. capsulatus RCs, replacement of the histidine ligand of the M-side bacteriochlorophyll a (BChla) molecule in the special pair primary-electron donor (denoted P) with a noncoordinating side chain leads to loss of the central Mg atom, converting it to a bacteriopheophytin a (BPheoa). This RC, the primary electron donor of which consists of one BChla (DL) and one BPheoa (DM), has been called the heterodimer mutant; the symbol D is used for P in this dimer (2, 5). In contrast to P. in which the lowest singlet electronic absorption band is relatively narrow (full width at half-maximum =500 cm-'), the absorption spectrum of D appears rather featureless and extends from =800-1000 nm. The quantum yield for the initial charge separation step in the heterodimer mutant is =50% of that in wild-type RCs; the remaining 50o of excited RCs decay rapidly to the ground state (5). The mechanism of charge separation in the heterodimer mutant has been suggested (5) to be the following:where HL is the BPheoa electron acceptor. [The notation *D is used to denote the excited state formed upon excitation at 870 nm under conditions ofthe experiments described in refs. 5 and 6. Internal charge transfer (CT) states of D are written as DL+DM-because BPheoa is "-300 meV easier to reduce than BChla (5-7). The hole on D+ may localize on the BChla (DL) half of the ...

The First Catalytic Step of the Light-driven Enzyme Protochlorophyllide Oxidoreductase Proceeds via a Charge Transfer Complex

“…If this is the case, large values of ⌬ and/or of Tr(⌬␣) are expected (23), similar to those observed for bacterial reaction center (21,22) and light harvesting complex I (27).…”

“…The unique presence of a chlorophyll-like molecule in the active site of POR allows techniques such as EPR (18,19), electron nuclear double resonance (ENDOR) (20), and Stark spectroscopies (21)(22)(23), which have proven to be invaluable for the analysis of photosynthetic systems, to be used to follow catalytic events in an enzyme reaction for the first time. Consequently, in the present work we have used a combination of these approaches in conjunction with low temperature absorbance spectroscopy to characterize the A 696 intermediate and show that the photochemical step involves hydride transfer from the NADPH molecule to form a charge transfer complex.…”

Excited-State Electronic Asymmetry of the Special Pair in Photosynthetic Reaction Center Mutants: Absorption and Stark Spectroscopy

“…In the M202HL/M160LH mutant, the absorption spectrum of the dimer becomes even broader than that in M202HL. Interestingly, the higher-energy feature around 860 nm is more intense than the lower energy feature around 940 nm in the M202HL/M160LH mutant, whereas the two features have comparable intensities in the M-side heterodimer (10). In M202HL/L131LH, where a hydrogen bond is added to the L-side of the dimer ( Figure 5, center panel), the dimer absorption band is narrower than that in M202HL, though it is still broader than in WT.…”

“…If either Mg-ligating histidine residue is replaced with Leu, a heterodimer is formed in which one of the BChls is replaced by a bacteriopheophytin (BChl in which the Mg is replaced by two hydrogen atoms) (9). The special pairs in these RCs have significantly altered spectroscopic (10,11) and functional properties (12). Mutants have been designed in which the histidine residue (His L168) that is hydrogen-bonded to the peripheral conjugated carbonyl group is removed or in which non-hydrogen-bonding amino acids near to conjugated carbonyl groups of P (residues M160, L131, and M197) are replaced by histidine residues that are capable of forming hydrogen bonds (13)(14)(15)(16).…”

Mg Coordination by Amino Acid Side Chains Is Not Required for Assembly and Function of the Special Pair in Bacterial Photosynthetic Reaction Centers

“…The absorption spectrum alone, even at cryogenic temperatures, does not provide definitive information on pigment content, the classic case being borohydride-treated RCs (Struck et al, 1991), and to a lesser extent the "reverse heterodimer" mutant, (L)H173L . However, the Stark effect spectrum is extremely sensitive to changes in the nature of the pigments comprising P. The Stark effect for the lowest energy electronic transition of P is dramatically larger in both the normal (M)H202L (Hammes et al, 1990) and reverse (L)-H173L heterodimers of Rb. sphaeroides.…”