Search

Cytochrome bd-I is one of the three proton motive force-generating quinol oxidases in the\ud O2-dependent respiratory chain of Escherichia coli. It contains one low-spin haem (b558)\ud and the two high-spin haems (b595 and d) as the redox-active cofactors. In order to examine\ud the flash-induced intraprotein reverse electron transfer (the so-called ''electron backflow''),\ud CO was photolyzed from the ferrous haem d in one-electron reduced (b558\ud 3+b595\ud 3+d2+-CO)\ud cytochrome bd-I, and the fully reduced (b5582+b5952+d2+-CO) oxidase as a control. In contrast\ud to the fully reduced cytochrome bd-I, the transient spectrum of one-electron reduced\ud oxidase at a delay time of 1.5 μs is clearly different from that at a delay time of 200 ns. The\ud difference between the two spectra can be modeled as the electron transfer from haem d to\ud haem b595 in 3–4% of the cytochrome bd-I population. Thus, the interhaem electron backflow\ud reaction induced by photodissociation of CO from haem d in one-electron reduced\ud cytochrome bd-I comprises two kinetically different phases: the previously unnoticed fast\ud electron transfer from haem d to haem b595 within 0.2–1.5 μs and the slower well-defined\ud electron equilibration with τ ~16 μs. The major new finding of this work is the lack of electron\ud transfer at 200 ns.

International audience; no abstract

The kinetics of electron transfer from phyllosemiquinone (PhQ(*-)) to the iron sulfur cluster F(X) in Photosystem I (PS I) are described by lifetimes of approximately 20 and approximately 250 ns. These two rates are attributed to reactions involving the quinones bound primarily by the PsaB (PhQ(B)) and PsaA (PhQ(A)) subunits, respectively. The factors leading to a approximately 10-fold difference between the observed lifetimes are not yet clear. The peptide nitrogen of conserved residues PsaA-Leu722 and PsaB-Leu706 is involved in asymmetric hydrogen-bonding to PhQ(A) and PhQ(B), respectively. Upon mutation of these residues in PS I of the green alga, Chlamydomonas reinhardtii , we observe an acceleration of the oxidation kinetics of the PhQ(*-) interacting with the targeted residue: from approximately 255 to approximately 180 ns in PsaA-L722Y/T and from approximately 24 to approximately 10 ns in PsaB-L706Y. The acceleration of the kinetics in the mutants is consistent with a perturbation of the H-bond, destabilizing the PhQ(*-) state, and increasing the driving force of its oxidation. Surprisingly, the relative amplitudes of the phases reflecting PhQ(A)(*-) and PhQ(B)(*-) oxidation were also affected by these mutations: the apparent PhQ(A)(*-)/PhQ(B)(*-) ratio is shifted from 0.65:0.35 in wild-type reaction centers to 0.5:0.5 in PsaA-L722Y/T and to 0.8:0.2 in PsaB-L706Y. The most consistent account for all these observations involves considering reversibility of oxidation of PhQ(A)(*-) and PhQ(B)(*-) by F(X), and asymmetry in the driving forces for these electron transfer reactions, which in turn leads to F(x)-mediated interquinone electron transfer.

International audience; Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property in the regulation of respiration [1]. We have previously shown that recombination kinetics of reduced CcO -NO from P. denitrificans on the picosecond time scale depend strongly on the NO:enzyme stoichiometry and inferred that more than one NO can be accomodated by the active site [2], already at mildly suprastoichimetric NO concentrations. We have now largely extended these studies by studying rebinding dynamics from the picosecond up to the microsecond time scale, by performing parallel steady-state EPR characterizations under similar conditions as the optical experiments, and comparing them with molecular modeling results. A comparative study was performed on CcO ba3 from T. thermophilus, where two NO molecules cannot be copresent in the active site in steady state due to its NO reductase activity [3]. The kinetic results allow to discriminate between different models of NOdependent recombination and show that the overall NO-escape probability out of the protein is high when only one NO is bound to aa3, whereas strong rebinding on the 15-ns time scale was observed for ba3. The EPR characterizations show similar results for aa3 at substoichiometries NO:enzyme ratios and for ba3, indicating formation of a six-coordinate heme-NO complex. The presence of a second NO molecule in the aa3 active site strongly modifies the heme-NO EPR spectrum, and can be rationilized by a rotation of the NO with respect to the proximal histidine. This proposal is consistent with molecular modeling studies. The binding properties of the second NO molecule will also be discussed.

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light- harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtii—locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b6f complex (A-AUU)—and measured electron flow though the cytochrome b6f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause–effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow