A reaction-induced FT-IR study of cyanobacterial photosystem I.

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.

Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels.

The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated K+ channels encodes molecular determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels. Crystal structures of T1 tetramers from Shaw and Shaker subfamilies reveal a common four-layered scaffolding. Within layer 4, on the hypothetical membrane-facing side of the tetramer, the Shaw T1 tetramer contains four zinc ions; each is coordinated by a histidine and two cysteines from one monomer and by one cysteine from an adjacent monomer. The amino acids involved in coordinating the Zn2+ ion occur in a HX5CX20CC sequence motif that is highly conserved among all Shab, Shaw and Shal subfamily members, but is not found in Shaker subfamily members. We demonstrate by coimmunoprecipitation that a few characteristic residues in the subunit interface are crucial for subfamily-specific tetramerization of the T1 domains.

A difference Fourier-transform infrared study of two redox-active tyrosine residues in photosystem II.

Photosystem II, the photosynthetic water-oxidizing complex, contains two redox-active tyrosine residues. Although current models suggest that these tyrosines are located in symmetric positions in the reaction center, there are functional differences between them. To elucidate those structural factors that give rise to this functional asymmetry, we have used difference Fourier-transform infrared spectroscopy to obtain the vibrational difference spectrum associated with the oxidation of each of these redox-active residues. Isotopic labeling was employed to definitively assign vibrational lines to the redox-active tyrosines. This work has shown that the vibrational spectra of the two redox-active species are significantly different from each other. This result suggests that the structure of the redox-active residue helps to determine its role in electron transfer in the reaction center.

Removal of stable tyrosine radical D+ affects the structure or redox properties of tyrosine Z in manganese-depleted photosystem II particles from Synechocystis 6803.

Photosystem II contains two redox-active tyrosines, D and Z. To understand the function of the dark stable tyrosine radical, D+, we have characterized two site-directed mutations at the D tyrosine residue in the transformable cyanobacterium, Synechocystis sp. PCC 6803, through the use of purified photosystem II particles (Noren, G. H., Boerner, R. J., and Barry, B. A. (1991) Biochemistry 30, 3943-3950). In manganese-depleted mutant particles, a light-induced EPR signal is observed. This signal contains a stable component, due to a chlorophyll radical, and an unstable component. The lineshape of the unstable, oxidized component, which we call M+, is obtained by subtraction; it has a lineshape different from tyrosine Z+/D+ and a g value of 2.004. Up to one M+ spin per reaction center can be photooxidized. The characteristic light-induced EPR signal ascribed to Z+ is not detected; under the same conditions, Z+ is detected in control preparations. The M+ radical lineshape is similar to the light-induced photosystem II radical identified in a site-directed mutant in the D1 polypeptide (YF161D1) (Noren, G. H., and Barry, B. A. (1992) Biochemistry 31, 3335-3342). Optical measurements on manganese-depleted photosystem II particles from control and D2 mutant preparations show that charge recombination kinetics between Q-A and an oxidized redox-active component are similar, to within a factor of two, in all three preparations. We conclude that lack of the stable tyrosine D+ alters the structure or redox properties of tyrosine Z in manganese-depleted preparations.