X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex

Abstract

The first steps of the global process of photosynthesis take place in specialized membrane pigment–protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment–protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (E m P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Å shift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the E m P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

Properties and Crystal Structure of the Cereibacter sphaeroides Photosynthetic Reaction Center with Double Amino Acid Substitution I(L177)H + F(M197)H

“…Analysis of the RC I(L177)H + F(M197)H structure in the vicinity of P B ( Figure 6 ) shows that the main pattern of the previously described H-bonding network, which includes the C2-acetyl carbonyl group of BChl P B , the imidazole group of His-M197, side groups of Asn-M195, Ser-L158, Asp-L155, and Tyr-M198 and two water molecules (assigned as waters-C and - E in Figure 6 and [ 10 ]), is similar to that observed in the RC with the single substitution F(M197)H. The sigma level of the electron density map peak for the water-C is low (1.7) and not sufficient to assert its presence with confidence. However, this water is observed in the structures of the wild-type RC and RC F(M197)H [ 10 ] as part of their H-bonding networks. As shown in Figure 6 , there is a high degree of probability that water-C is also present in the structure of the double mutant RC.…”

“…In both complexes, the imidazole group of His-M197 is located within the H-bond distance from the C2-acetyl carbonyl group of BChl P B . It was noted that the C2-acetyl group shows 29.7° out of plane rotation ( Figure 5 ), which is more significant compared with that observed in RC F(M197)H (20.7°) [ 10 ]. The data are consistent with spectral properties of RC I(L177)H + F(M197)H (as discussed below).…”

“…It has been shown that in the wild-type RC of C. sphaeroides, an H-bonding network also presents on the periplasmic side of BChl P A [ 10 ]. The network includes the C2-acetyl carbonyl group of BChl P A , the imidazole group of His-L168, side groups of Asn-L166, Asp-M184, and Tyr-L169 and a water molecule assigned as water-D.…”

“…Based on the recently resolved crystal structure of the RC F(M197)H, it was found that the increased stability of this complex is associated with the incorporation of the C2-acetyl carbonyl group of BChl P B into the H-bonding network surrounding this cofactor. It was also suggested that the size and pattern of the H-bonding networks near P can affect the oxidation potential of the BChl dimer [ 10 ]. However, more experimental data are needed to support this assumption.…”

Stabilization of Cereibacter sphaeroides Photosynthetic Reaction Center by the Introduction of Disulfide Bonds

“…H-bond networks have been shown to play an important role in the stability of reaction centers [ 8 ]. For C. sphaeroides RC, the H-bond of the acetyl group of each of the bacteriochlorophylls in the dimer ensures the stability of the complex at elevated temperature and pressure [ 9 , 10 ].…”