Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Abstract

Low-frequency (90-435 cm(-1)) NIR-excitation (875-900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421-2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P(M) in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D(2)O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P(M) in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P(M) in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH(2) stretching vibration is tentatively assigned to a band at 318 cm(-1), a frequency higher than that of the Mg-His stretch of the native pigment ( approximately approximately 235 cm(-1)). (3) The BChl core structure of P(M) in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D(2)O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.

In Situ, Protein-Mediated Generation of a Photochemically Active Chlorophyll Analogue in a Mutant Bacterial Photosynthetic Reaction Center

“…Water is suggested to be a ligand 10 as supported by an isotope effect on the Mg−water vibration. 11 In summary, a combination of amino acid and overall binding site characteristics likely predisposes the various pigment sites in the RC to incorporation/retention of the native BChl vs BPh or vice versa.…”

“…Typically, His serves as an axial ligand to the Mg of BChl molecules in RCs. Early work showed that when either His residue that ligates the Mg 2+ of a macrocycle of P is changed to Leu, P becomes a BPh–BChl heterodimer. , Similarly, replacing the axial His B B ligand with Leu results in a BPh (φ B ) at that site in both R. sphaeroides and R. capsulatus . , An analogous replacement with Leu of the axial His ligand of B A results in BPh at the site in Blastochloris viridis but not R. sphaeroides . , Surprisingly, when the His ligands to P’s macrocycles are changed to Gly, the two BChls of P are retained, possibly ligated with water molecules in this so-called “cavity” mutant of R. sphaeroides . , Remarkably, in a triple mutant that contains these same two His → Gly replacements and a third that removes a hydrogen bond to one 3-acetyl group of the dimer, the two BChl molecules comprising P are completely eliminated yet the remainder of the RC appears intact . Replacing an appropriately positioned Leu near either H A or H B with His results in incorporation of a BChl denoted β A or β B , respectively. − Mutant RCs in which B A , , H A , or H B is absent have also been reported.…”

“…8,9 Surprisingly, when the His ligands to P's macrocycles are changed to Gly, the two BChls of P are retained, possibly ligated with water molecules in this so-called "cavity" mutant of R. sphaeroides. 10,11 Remarkably, in a triple mutant that contains these same two His → Gly replacements and a third that removes a hydrogen bond to one 3-acetyl group of the dimer, the two BChl molecules comprising P are completely eliminated yet the remainder of the RC appears intact. 12 Replacing an appropriately positioned Leu near either H A or H B with His results in incorporation of a BChl denoted β A or β B , respectively.…”