Marten H. Vos

Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 Å, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH•) induces an electron transfer over ≈15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH• ← W382 ← W359 ← W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH• decayed with a time constant τ ≈ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (τ ≈ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH• is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (≈0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH• is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH• (τ ≈ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH• in WT and from W359 to excited FADH• in W382F mutant photolyase were estimated and related to the donor–acceptor distances.

Femtosecond processes in proteins

4. Photosynthetic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4.1. Vibrational coherence in bacterial reaction centers . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4.2. Characteristics of low-frequency vibrations in photosynthetic proteins . . . . . . . . . . . . . 7 4.3. Vibrational coherence and energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.4. Electron transfer in bacterial reaction centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.5. Energy transfer in LH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Coherent reaction dynamics in a bacterial cytochrome c oxidase

Biological reactions in protein complexes involve structural dynamics spanning many orders of magnitude in time. In standard descriptions of catalysis by enzymes, the transition state between reactant and product is reached by thermal, stochastic motion. In the ultrashort time domain, however, the protein moiety and cofactor motions leading to altered conformations can be coherent rather than stochastic in nature. Such coherent motions may play a key role in controlling the accessibility of the transition state and explain the high efficiency of the reaction. Here we present evidence for coherent population transfer to the product state during an ultrafast reaction catalysed by a key enzyme in aerobic organisms. Using the enzyme cytochrome c oxidase aa3 from the bacterium Paracoccus denitrificans, we have studied haem dynamics during the photo-initiated ultrafast transfer of carbon monoxide from haem a3 to CuB by femtosecond spectroscopy. The ground state of the unliganded a3 species is populated in a stepwise manner in time, indicating that the reaction is mainly governed by coherent vibrations (47 cm-1). The reaction coordinate involves conformational relaxation of the haem group and we suggest that ligand transfer also contributes.

Interaction of Carbon Monoxide with the Apoptosis-Inducing Cytochrome c-Cardiolipin Complex

The interaction of mitochondrial cytochrome (cyt) c with cardiolipin (CL) is involved in the initial stages of apoptosis. This interaction can lead to destabilization of the heme-Met80 bond and peroxidase activity [Basova, L. V., et al. (2007) Biochemistry 46, 3423-3434]. We show that under these conditions carbon monoxide (CO) binds to cyt c, with very high affinity ( approximately 5 x 10(7) M(-1)), in contrast to the native cyt c protein involved in respiratory electron shuttling that does not bind CO. Binding of CO to the cyt c-CL complex inhibits its peroxidase activity. Photodissociated CO from the cyt c-CL complex shows <20% picosecond geminate rebinding and predominantly bimolecular rebinding, with a second-order rate constant of approximately 10(7) M(-1) s(-1), an order of magnitude higher than in myoglobin. These findings contrast with those of Met80X mutant cyt c, where picosecond geminate recombination dominates due to the rigidity of the protein. Our data imply that CL leads to substantial changes in protein conformation and flexibility, allowing access of ligands to the heme. Together with the findings that (a) approximately 30 CL per cyt c are required for full CO binding and (b) salt-induced dissociation indicates that the two negative headgroup charges interact with approximately 5 positive surface charges of the protein, these results are consistent with a CL anchorage model with an acyl chain impaled in the protein [Kalanxhi, E., and Wallace, C. J. A. (2007) Biochem. J. 407, 179-187]. The affinity of CO for the complex is high enough to envisage an antiapoptotic effect of nanomolar CO concentrations via inhibition of the cyt c peroxidase activity.

Ultrafast ligand rebinding in the heme domain of the oxygen sensors FixL and Dos: General regulatory implications for heme-based sensors

Heme-based oxygen sensors are part of ligand-specific two-component regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate. To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy to study ligand dynamics in the heme domains of the oxygen sensors FixL from Bradyrhizobium japonicum (FixLH) and Dos from Escherichia coli (DosH). The heme coordination with different ligands and the corresponding ground-state heme spectra of FixLH are similar to myoglobin (Mb). After photodissociation, the excited-state properties and ligand-rebinding kinetics are qualitatively similar for FixLH and Mb for CO and NO as ligands. In contrast to Mb, the transient spectra of FixLH after photodissociation of ligands are distorted compared with the ground-state difference spectra, indicating differences in the heme environment with respect to the unliganded state. This distortion is particularly marked for O2. Strikingly, heme–O2 recombination occurs with efficiency unprecedented for heme proteins, in ≈5 ps for ≈90% of the dissociated O2. For DosH–O2, which shows 60% sequence similarity to FixLH, but where signal detection and transmission presumably are quite different, a similarly fast recombination was found with an even higher yield. Altogether these results indicate that in these sensors the heme pocket acts as a ligand-specific trap. The general implications for the functioning of heme-based ligand sensors are discussed in the light of recent studies on heme-based NO and CO sensors.

An electroluminescence study of stabilization reactions in the oxygen-evolving complex of Photosystem II**

The stabilization of the photosynthetic charge separation in Photosystem II by secondary reactions was studied using chlorophyll luminescence induced by electric field pulses in a suspension of preilluminated osmotically swollen chloroplasts. This ‘electroluminescence’ was measured as a function of the delay time between illumination and field pulse, and as a function of the number of preilluminating flashes. The result is a survey of, in principle, all stabilization and deactivation processes beyond the state Z + Q − A , which is formed within the approx. 20 μs time resolution of the method. Most of these could be identified with known secondary electron transfer reactions. A 20-fold stabilization with a half-time of 330 μs is attributed to Q − A reoxidation. No further stabilization at the acceptor side seemed to occur and no flash number dependence was detected, although a normal Q B /Q − B oscillation was found in ultraviolet absorbance. With regard to the donor side, the data are consistent with the known S-state-dependent Z + reduction times and indicate values of 9, 5 and 65 for the equilibrium constants associated with this reaction on the transitions S 1 →S 2 , S 2 →S 3 and S 3 →S 0 (O 2 ) respectively. Z + reduction was found to be electrogenic and exposed to about 5% of the membrane potential. An 0.1 s phase in S 0 is attributed to oxygen release. S 2 and S 3 are further stabilized in two phases of unknown origin with half-times of 15 ms and 0.4 s, followed by a final 20 s phase attributed to deactivation. In S 1 , Z + reduction was probably hidden in an unresolved fast phase present on all transitions, but in addition a 350 μs phase was found, which might be related to proton release. In nearly 20% of the Photosystem II reaction centers electron transfer beyond Q − A was inhibited. In these centers Z + reduction appeared to take about 1 ms and charge recombination followed in phases of about 8, 80 and 800 ms half-time.

Discovery of Intracellular Heme-binding Protein HrtR, Which Controls Heme Efflux by the Conserved HrtB-HrtA Transporter in Lactococcus lactis

Background: Heme is an essential cofactor yet toxic in free form, necessitating strict intracellular control. Results: A heme sensor regulates the conserved hrtBA genes in Lactococcus lactis, whose products mediate heme efflux. Conclusion: L. lactis controls heme homeostasis by sensing intracellular heme and activating heme efflux. Significance: The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis. Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.

Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding.

Vibrational coherence in bacterial reaction centers: spectroscopic characterisation of motions active during primary electron transfer

Abstract Coherent dynamics during the primary electron transport reaction in bacterial reaction centers was studied. We analyzed the spectral dependence of oscillatory features in multicolour optical pump–probe measurements in the spectral region of both the stimulated emission of the P * reactant state and the absorption of the bacteriochlorophyll monomers. In the latter region, the manifold of activated modes gives rise to a very complex oscillatory pattern. Using simple schemes of spectroscopic probing of wave packet dynamics on the P * potential energy surface, the origin of the coherences is discussed. In addition to transitions probing P * directly, electrochromic shifts of the B bands induced by oscillating charges may contribute to the signal.