Characterization of Site-Directed Mutants in the Q(B) Binding Site of the Photosynthetic Reaction Center of Rhodobacter Sphaeroides

Takahashi, Eiji

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Description

Title

Characterization of Site-Directed Mutants in the Q(B) Binding Site of the Photosynthetic Reaction Center of Rhodobacter Sphaeroides

Author(s)

Takahashi, Eiji

Issue Date

1992

Doctoral Committee Chair(s)

Wraight, Colin A.

Department of Study

Plant Biology

Discipline

Plant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Biology, Molecular
• Chemistry, Biochemistry
• Biophysics, General

Abstract

• Protonation and electron transfer events in reaction centers (RCs) from photosynthetic purple bacterium Rhodobacter sphaeroides were investigated by characterization of site-directed mutants. The RC deletion mutant GaKM(+) was constructed in the Ga strain of Rb. sphaeroides and then complemented in trans by mutant RC genes to produce mutant RC proteins.
• In the mutant L212EQ, the pH dependences of the one electron $\rm Q\sb{A}\sp-Q\sb{B} \leftrightarrow Q\sb{A}Q\sb{B}\sp-$ equilibrium and electron transfer rate at alkaline pH, seen for the wild type (Wt) RCs, were essentially eliminated, as also reported by Paddock et al. (1989). Thus, Glu$\sp{\rm L212},$ with an unusually high pK $(\sim$9.6), inhibits the electron transfer when ionized. The L212EQ RC also exhibited a significantly reduced rate of transfer of the second proton to Q$\sb{\rm B}\sp{2-},$ resulting in substantially lower maximum steady state rate of cytochrome c photooxidation when compared to the Wt.
• The mutation L213DN resulted in even more dramatic effects, including a large increase in the $\rm Q\sb{A}\sp-Q\sb{B}\leftrightarrow Q\sb{A}Q\sb{B}\sp-$ equilibrium in favor of Q$\sb{\rm B}$ reduction and an altered pH dependence of the equilibrium. Transfer of the second electron to Q$\sb{\rm B}\sp-$ was drastically affected.
• Comparison of the behavior of Wt and the three mutant RC types leads to the following conclusions: the pK of Asp$\sp{\rm L213}$ in the Wt is $\approx$4 for the Q$\sb{\rm A}$Q$\sb{\rm B}$ state (p$K\sb{\rm Q\sb{B}})$ and $\approx$5 for the Q$\sb{\rm A}$Q$\sb{\rm B}\sp-$ state (p$K\sb{\rm Q\sb{B\sp-}});$ for Glu$\sp{\rm L212},$ p$K\sb{\rm Q\sb{B}}$ $\approx$ 9.5 and p$K\sb{\rm Q\sb{B\sp-}}$ $\approx$ 11. In L213DN mutant RCs, p$K\sb{\rm Q\sb{B}}$ of Glu$\sp{\rm L212}$ is $\leq$7, indicating that the high pK values of Glu$\sp{\rm L212}$ in the Wt are due largely to electrostatic interaction with the ionized Asp$\sp{\rm L213}.$ Transfer of the second electron and all associated proton uptake to form $\rm Q\sb{B}H\sb2$ is drastically inhibited in double mutant and L213DN mutant RCs.
• This work shows a very high degree of specificity for the residues involved in the terminal steps of proton donation to Q$\sb{\rm B}$ in Rb. sphaeroides. However, it is evident from sequence comparisons with other bacterial species that other configurations can achieve the same end. The Q$\sb{\rm B}$ site may therefore be viewed as a proton impermeable structure into which protons may be transferred via one or two specific ionizable residues which may be located at a variety of positions, depending on species or strain, i.e. on the specific amino acid sequence. (Abstract shortened by UMI.)

Graduation Semester

1992

Type of Resource

text

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