First site-specific incorporation of a noncanonical amino acid into the photosynthetic oxygen-evolving complex.
In photosystem II (PSII), water is oxidized at the oxygen-evolving complex. This process occurs through a light-induced cycle that produces oxygen and protons. While coupled proton and electron transfer reactions play an important role in PSII and other proteins, direct detection of internal proton transfer reactions is challenging. Here, we demonstrate that the unnatural amino acid, 7-azatryptophan (7AW), has unique, pH-sensitive vibrational frequencies, which are sensitive markers of proton transfer. The intrinsically disordered, PSII subunit, PsbO, which contains a single W residue (Trp241), was engineered to contain 7AW at position 241. Fluorescence shows that 7AW-241 is buried in a hydrophobic environment. Reconstitution of 7AW(241)PsbO to PSII had no significant impact on oxygen evolution activity or flash-dependent protein dynamics. We conclude that directed substitution of 7AW into other structural domains is likely to provide a nonperturbative spectroscopic probe, which can be used to define internal proton pathways in PsbO.
Department of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology , 901 Atlantic Drive NW, Atlanta, Georgia 30332, United States.
Adam R
AR
Offenbacher
Cynthia V
CV
Pagba
Brandon C
BC
Polander
Udita
U
Brahmachari
Bridgette A
BA
Barry
D000596:Amino Acids / Q000737:chemistry*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D015394:Molecular Structure; D010100:Oxygen; D045332:Photosystem II Protein Complex / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014867:Water / Q000737:chemistry
D000596:Amino Acids; D045332:Photosystem II Protein Complex; D014867:Water; C006657:7-azatryptophan; D014364:Tryptophan; D010100:Oxygen