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

Abstract

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (H B ) in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (C B ) in place of H B . The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C−C bond in ring D and a CC bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a. In such RCs, charge-separated state P + C B − forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a) denoted β B in place of H B .Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% β B in the H B site, while most other amino acids result in RCs that retain H B (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides

“…The methods for protein engineering, cell growth and RC purification have been described recently. 32 In brief, R. sphaeroides mutant RCs were constructed using a cassette-based mutagenesis platform based on engineered plasmid pBBRRWsLsMα − carrying genes of the puf operon. 19 Synthetic oligonucleotide cassettes were designed to contain the desired substitution and were ligated to derivatives of plasmid pBBRRWsLsMα − carrying the desired background substitutions.…”

“…The spectral/temporal data matrices were analyzed as described previously. 32,33 For each mutant RC, the yield of ET from P* to the B-side cofactors to produce P + H B − was determined via TA spectroscopy. The method (shown in Figure 3 and described previously 6 ) is based on comparison to the ∼100% yield of ET from P* to H A in the W(M252)V mutant (denoted WT V ).…”

“…Residue LeuL185 is positioned over one face of H B and is C 2 -symmetry related to the native LeuM214 similarly centered over one face of H A (Figure 2). Recently, we reported the results of substitution of all native amino acids for the native Leu at L185 in the bg1 and bg2 backgrounds on RC pigment content and on the rates and yields of ET at 295 K. 32 Results on the Trp-bg2 mutant at 77 K are compared here with those for Trp-YFH RC, which lacks the T(M133)E substitution. The two mutants are defined in Table 1 and depicted in Figure 9b and a, respectively.…”

“…Saturation mutagenesis of the L185 site 32 (native residue a Leu) results in incorporation of a BChl denoted β B in place of H B , for amino acids His, Asp, Asn, or Gln. When L185 is Glu, an unusual chlorin identified as 17,18-didehydro BChl a is incorporated in the H B site in 45% of the RCs and BChl (β B ) in 50%, while 5% retain the native BPh (H B ).…”

“…6 Trp substitution at L185 provides relatively high yield (∼80%) of P + H B − at 295 K that is comparable to that for the bg1 and bg2 "backgrounds" (∼75%). 32 Native amino acids shown in black are replaced by residues given by the single letter codes (orange). These mutations are defined in Table 1.…”