The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B).
In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the hundreds of microseconds range) and fast phase (microseconds to tens of microseconds range) in AM260(W-->C) RCs. We conclude that the unusually strong hydrogen bond between the carbonyl of QA and His M219 in the Rb. sphaeroides RC is not obligatory for efficient electron transfer from QA- to QB.
Breton
Jacques
J
Service de Bioénergétique, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France, UMR 6191 CNRS-CEA-Aix-Marseille II, DEVM-CEA-Cadarache, 13108 St Paul lez Durance, France. cadara3@dsvidf.cea.fr
Lavergne
Jérôme
J
Wakeham
Marion C
MC
Nabedryk
Eliane
E
Jones
Michael R
MR
D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004579:Electron Transport / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011809:Quinones / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water
D045322:Photosynthetic Reaction Center Complex Proteins; D011809:Quinones; D014867:Water; D006639:Histidine; D007501:Iron; D003545:Cysteine