FTIR Spectroscopy of Synechocystis 6803 Mutants Affected on the Hydrogen Bonds to the Carbonyl Groups of the PsaA Chlorophyll of P700 Supports an Extensive Delocalization of the Charge in P700

• Published in: Biochemistry (Easton) Vol. 43; no. 26; pp. 8380 - 8390
• Main Authors: Pantelidou, Maria, Chitnis, Parag R, Breton, Jacques
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06-07-2004
• Subjects: Adhesins, Bacterial; Bacterial Proteins; Carbon - chemistry; Carrier Proteins - chemistry; Cell Division; Chlorophyll - chemistry; Cyanobacteria - metabolism; Hydrogen Bonding; Light; Lipoproteins - chemistry; Membrane Transport Proteins; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Nitrogen - chemistry; Spectroscopy, Fourier Transform Infrared - methods; Threonine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049515j

P700, the primary electron donor of photosystem I is an asymmetric dimer made of one molecule of chlorophyll a‘ (PA) and one of chlorophyll a (PB). While the carbonyl groups of PA are involved in hydrogen-bonding interactions with several surrounding amino acid side chains and a water molecule, PB does not engage in hydrogen bonding with the protein. Light-induced FTIR difference spectroscopy of the photooxidation of P700 has been combined with site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the influence of these hydrogen bonds on the structure of P700 and P700+. When the residue Thr A739, which donates a hydrogen bond to the 9-keto CO group of PA, is changed to Phe, a differential signal at 1653(+)/1638(−) cm-1 in the P700+/P700 FTIR difference spectrum upshifts by ∼30−40 cm-1, as expected for the rupture of the hydrogen bond or, at least, a strong decrease of its strength. The same upshift is also observed in the FTIR spectrum of a triple mutant in which the residues involved in the three main hydrogen bonds to the 9-keto and 10a-carbomethoxy groups of PA have been changed to the symmetry-related side chains present around PB. In addition, the spectrum of the triple mutant shows a decrease of a differential signal around 1735 cm-1 and the appearance of a new signal around 1760 cm-1. This is consistent with the perturbation of a bound 10a-ester CO group that becomes free in the triple mutant. All of these observations support the assignment scheme proposed previously for the carbonyls of P700 and P700+ [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585−11592] and therefore reinforce our previous conclusions that the positive charge in P700+ is largely delocalized over PA and PB.