CO Photolysis Research Articles

Electrogenic events upon photolysis of CO from fully reduced cytochrome c oxidase

CO photolysis from fully reduced Paracoccus denitrificans aa3-type cytochrome c oxidase in the absence of O2 was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5μs) phase was followed by slower potential generation with a time constant varying from 8μs at pH 7 to 250μs at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10–30%. The amplitude of the fast phase corresponds to charge movement of 0.83Å perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of CcO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.

Vibrational Coherence Spectroscopy of the Heme Domain in the CO-Sensing Transcriptional Activator CooA

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here, we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants, and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm(-1) in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole-bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm(-1) in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm(-1) that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm(-1)). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm(-1) in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm(-1) disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated, and although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.

Evidence for the Presence of Two Conformations of the Heme a3-CuB Pocket of Cytochrome caa3 from Thermus thermophilus

Resonance Raman (RR) and "light" minus "dark" Fourier transform infrared (FTIR) difference spectra are reported for the CO-bound caa(3) oxidase from Thermus thermophilus. Two Fe-CO stretching modes at 518 and 507 cm(-1), the Fe-C-O bending mode at 570 cm(-1), and three C-O modes of heme a(3) at 1958, 1967, and 1973 cm(-1) have been identified in the RR and FTIR spectra, respectively. The FTIR "light" minus "dark" spectrum indicates the formation of Cu(B)CO as revealed by its ν(CO) at 2060/2065 cm(-1). We assign the bands at 518 (ν(Fe-CO)) and 1967/1973 cm(-1) (ν(C-O)) as the α-conformation. We also assign the bands at 507 and 1958 cm(-1) (ν(C-O)) as originating from the β-conformation of the enzyme. A frequency upshift of the heme a(3) Fe-His mode is observed subsequent to CO photolysis from 209 cm(-1) in the equilibrium deoxy enzyme to 214 cm(-1) in the photoproduct. The caa(3) data, distinctly different from those of ba(3) oxidase, are discussed in terms of the coupling of the α- and β-conformations that occur in heme-copper oxidases with catalytic function. The dynamics between the heme a(3) and heme a propionates as revealed by the perturbation of the bending vibrations δ(prop) of hemes a and a(3) at 385 and 392 cm(-1), respectively, induced upon CO binding to heme a(3) is discussed in terms of the protonic connectivity between the heme a ring-D propionate/Arg site with that of the heme a(3) ring-D propionate-H(2)O site that leads to the highly conserved in the heme-copper oxidases water pool.

Protein Folding Dynamics of Cytochrome c Seen by Transient Grating and Transient Absorption Spectroscopies

We investigate optically triggered protein folding dynamics of cytochrome c (Cytc) using transient grating (TG) and transient absorption (TA) spectroscopies. Despite many studies on protein folding dynamics of Cytc, a well-known model protein, direct spectroscopic evidence for the three-dimensional global folding process has been rarely reported. By measuring the TG signal of CO-bound Cytc (Cytc-CO) in the presence of a denaturant, we clearly detected the change of diffusion coefficient that reflects the size change of Cytc upon photodissociation of the CO ligand from unfolded Cytc-CO. The quantitative analysis of TG signals supports that the optically triggered folding reaction of Cytc in the presence of a denaturant takes place through a detectable intermediate (three-state folding kinetics). This is in contrast with the two-state folding dynamics of Cytc under a denaturant-free environment without any detectable intermediate. (1) From the quantitative global analysis of the TG signals, the rate constants for the U → I and I → N transitions in a CAPS buffer solution (pH 7) at room temperature in the presence of a denaturant at various concentrations are determined to be 1065 ± 17 to 3476 ± 103 s(-1) and 101 ± 6 to 589 ± 21 s(-1), respectively. In addition, the activation energies (E(a)) for the U → I and I → N transitions are determined to be 8.7 ± 1.0 kcal/mol and 7.1 ± 1.3 kcal/mol, respectively. The folding dynamics of Cytc initiated by the CO photolysis is discussed based in terms of the protein size change.

Cytoglobin conformations and disulfide bond formation

The oligomeric state and kinetics of ligand binding were measured for wild‐type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N‐ and C‐terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

Heme–heme and heme–ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b 558, b 595, and d. The role of heme b 595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d–CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b 595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b 595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b 595, and not that of heme b 558, controls the pathway(s) by which CO migrates between heme d and the medium.

Cytoglobin conformations and disulfide bond formation

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

Atmospheric chemistry on Venus, Earth, and Mars: Main features and comparison

This paper deals with two common problems and then considers major aspects of chemistry in the atmospheres of Mars and Venus. (1) The atmospheres of the terrestrial planets have similar origins but different evolutionary pathways because of the different masses and distances to the Sun. Venus lost its water by hydrodynamic escape, Earth lost CO 2 that formed carbonates and is strongly affected by life, Mars lost water in the reaction with iron and then most of the atmosphere by the intense meteorite impacts. (2) In spite of the higher solar radiation on Venus, its thermospheric temperatures are similar to those on Mars because of the greater gravity acceleration and the higher production of O by photolysis of CO 2. O stimulates cooling by the emission at 15 μm in the collisions with CO 2. (3) There is a great progress in the observations of photochemical tracers and minor constituents on Mars in the current decade. This progress is supported by progress in photochemical modeling, especially by photochemical GCMs. Main results in these areas are briefly discussed. The problem of methane presents the controversial aspects of its variations and origin. The reported variations of methane cannot be explained by the existing data on gas-phase and heterogeneous chemistry. The lack of current volcanism, SO 2, and warm spots on Mars favor the biological origin of methane. (4) Venus’ chemistry is rich and covers a wide range of temperatures and pressures and many species. Photochemical models for the middle atmosphere (58–112 km), for the nighttime atmosphere and night airglow at 80–130 km, and the kinetic model for the lower atmosphere are briefly discussed.

CO Binding and Ligand Discrimination in Human Myeloperoxidase

Despite the fact that ferrous myeloperoxidase (MPO) can bind both O(2) and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous-CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, k(on) and k(off) were 0.14 mM(-1) x s(-1) and 0.23 s(-1), respectively, yielding an unusually high K(D) of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O(2). The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pK(a) of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm(-1). Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pK(a) approximately 7.4) with the 1927 cm(-1) form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.

Role of His(E7) in Regulating Ligand Binding to the Subunits of Human HbA

To resolve previous discrepancies between structural and functional studies, the role of the distal histidine in HbA was re-evaluated by preparing Gly, Ala, Leu, Gln, Phe and Trp(E7) mutants and measuring the effects on O2, CO, and NO binding to mutant/wild type hybrid tetramers and isolated mutant subunits. Substituting His(E7) with apolar amino acids dramatically increases O2 dissociation (20-500-fold) in both subunits, suggesting equally strong hydrogen bonds between His(E7) and bound O2 (ΔGH-bond ≈ −5.6 kJ/mol). Increasing the size of the E7 residue from Gly to Phe results in monotonic decreases in the bimolecular rates of ligand binding to both subunits, supporting the E7 gate as the pathway for ligand entry in HbA. The results for the Trp(E7) mutants are more complex. Both fast (∼150-200 μM−1s−1) and one or more slow phases (1 to 0.1 μM−1s−1) are observed after photolysis of CO. The fraction of the fast phase decreases markedly when [CO] is lowered. In contrast, when isolated α and βTrp(E7) deoxyHb subunits are mixed with CO in stopped flow experiments, only slow phases are observed. Thus, after photolysis of the CO form of Trp(E7) mutants, there appears to be a competition between bimolecular ligand rebinding to an “open” conformation and the movement of the indole side chain back into the E7 channel forming an equilibrium “closed,” slowly reacting conformation. This mechanism is supported by the crystal structure of the CO form of α (wt)/βTrp(E7), in which the mutant indole side chain is in an open conformation exposed to solvent. In the deoxyHb crystal structure of αTrp(E7)/β (wt), the indole ring of Trp(E7) is in a closed conformation, blocking both the ligand binding site and the E7 channel for ligand entry.

1P107 1G1340 チトクロムc酸化酵素のCO光解離後の構造緩和 : ヘム側鎖のラマンバンドの帰属(ヘム蛋白質,口頭発表,第48回日本生物物理学会年会)

1P107 1G1340 チトクロムc酸化酵素のCO光解離後の構造緩和 : ヘム側鎖のラマンバンドの帰属(ヘム蛋白質,口頭発表,第48回日本生物物理学会年会)

Direct Observation of Ligand Dynamics in Cytochrome C Using Time-Resolved FTIR Spectroscopy

Horse heart cytochrome c (cyt c) has emerged as a paradigm for the study of protein folding. The folding of reduced cyt c induced by photodissociation of CO from the CO-bound unfolded protein has been studied extensively. Following a nanosecond light pulse, four transitions have been resolved with time constants of approximately 1-5, 50-100, 200-500, and 1000-10,000 μs. While originally thought to be associated with CO rebinding to two different partially folded states of cyt c, the two slower process are now understood to reflect the bimolecular reassociation of CO followed by religation of the His18, which by the base elimination mechanism is induced to dissociate after CO photolysis. Thus, the two slower time constants turn out not to report on protein folding, but instead reflect the complexity of heme ligation. The two faster time constants have been attributed to ligation at the heme center by protein side chains. Here, to unambiguously determine the post-photodissociation steps involving CO, we monitored the CO vibration following photodissociation with step-scan FT IR spectroscopy. We find that like the slower timescale processes, the 50-100 μs timescale process is associated not with protein dynamics, but with CO ligand dynamics. The data clearly demonstrate that whatever the origins of the spectral changes, they clearly involve CO rebinding or changes in the environment of an already bound CO ligand. In addition to these fast dynamics, we also find multi-phasic CO rebinding on timescales of 1-100 s. The dependence of the associated amplitudes on denaturant concentration suggests that a unique species exists at intermediate denaturant concentrations, consistent with a folding-unfolding process of the protein driven by CO dissociation. This may represent the first evidence for the long sought-after protein folding process triggered by photo-induced CO dissociation.

Protein Tertiary Structural Changes Visualized by Time-Resolved X-ray Solution Scattering

We obtained a solution structural model of myoglobin (Mb) formed upon the CO photolysis of MbCO by analyzing time-resolved X-ray solution scattering data. An experiment-restrained rigid-body molecular dynamics simulation was used to find the best model whose theoretical difference scattering curve gives a satisfactory agreement with the experimental data at the time delay of 10 ns. The obtained solution model shows structural changes similar to crystallographic models for MbCO --> Mb but also displays a noticeable difference in that the N-terminus and F helix show larger structural changes.

Photothermal studies of CO photodissociation from peroxidases from horseradish and soybean

In this work, the results of photoacoustic calorimetry (PAC) studies involving CO photodissociation from horseradish peroxidase (HRP) and soybean peroxidase (SBP) are discussed. Both proteins contain Fe-protoporphyrin IX active sites and relatively open distal heme pockets (i.e., direct solvent access). In addition, it has been shown previously that SBP binds a Tris molecule in the distal pocket near the heme group potentially regulating ligand binding to the heme iron. Results of PAC studies indicate a fast (< ∼ 50 ns) relaxation for both HRP and SBP subsequent to CO photolysis in both phosphate and Tris buffers and with varying concentrations of Tris. However, the molar volume/enthalpy changes associated with CO release are distinct between the two proteins. In the case of HRP, CO photolysis results in an enthalpy change of ∼ 2 kcal mol − 1 and volume change of ∼ − 12 mL mol − 1 attributed to solvation/structural changes regardless of buffer conditions. In contrast, SBP exhibits buffer and ionic strength dependent enthalpy changes ranging from ∼ − 23 kcal mol − 1 in 50 mM phosphate buffer to ∼ 6 kcal mol − 1 in Tris buffer with volume changes similar to those observed in HRP. The results are consistent with a model in which photodissociation of CO from ferrous HRP or SBP leads to CO migration from the distal heme pocket to the bulk solvent with a corresponding input of a water molecule all occurring in < ∼ 50 ns. The differences in enthalpies are attributed to variations in hydrogen bond formation between the incoming water molecule(s) and the protein matrix in both HRP and SBP.

Measurements of Heme Relaxation and Ligand Recombination in Strong Magnetic Fields

Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 T). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O(2)), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit. However, in the case of CO binding to heme in glycerol and O(2) binding to myoglobin, we observe a small magnetic field dependent change in the early time amplitude of the optical response that is assigned to heme cooling. One possibility, consistent with this observation, is that there is a weak magnetic field dependence of the nonradiative branching ratio into the vibrationally hot electronic ground state during CO photolysis. Ancillary studies of the "spin-forbidden" CO binding reaction in a variety of heme compounds in the absence of magnetic field demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO binding to heme is not always retarded by unfavorable spin selection rules involving a double spin-flip superexchange mechanism. In fact, it appears that the small prefactor ( approximately 10(9) s(-1)) found for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO rebinding. These results point to unresolved fundamental issues that underlie the theory of heme-ligand photolysis and rebinding.

Temperature dependence of isotopic fractionation in CO 2 photolysis

The effect of temperature on the heavy isotope ratios of the products (CO and O 2) of photolysis of CO 2 by ∼185 nm photons was studied. The isotope ratios 18O/ 16O and 17O/ 16O in O 2 and CO and 13C/ 12C in CO increase with temperature by ∼1‰ per K. The increase in 17O/ 16O in O 2 is related linearly to the increase in 18O/ 16O with a slope 0.8 contrary to the mass dependent value of 0.5. Variations in factors related to the transition dipole moment and Boltzmann distribution of the vibrational state populations are probably responsible for the observed change. Temperature dependence of the gas production rate agrees with the reported cross-section variations.

Isotopic records in CM hibonites: Implications for timescales of mixing of isotope reservoirs in the solar nebula

The magnesium isotopic compositions of 26 hibonite-bearing inclusions from the CM chondrite Murchison, as well as isotopic measurements on a subset of these samples for oxygen, titanium, and lithium–beryllium–boron are reported along with oxygen isotopic data for an additional 13 hibonites that were previously investigated for other isotope systems (magnesium, potassium, calcium, and titanium) and rare earth element concentrations. Magnesium isotopic compositions divide CM hibonites into two distinct populations which correlate perfectly with their mineralogy and morphology, as previously discovered by Ireland [Ireland T. R. (1988) Correlated morphological, chemical, and isotopic characteristics of hibonites from the Murchison carbonaceous chondrite. Geochim. Cosmochim. Acta 52, 2827–2839]: Spinel-HIBonite spherules (SHIBs) bear evidence of in situ 26Al decay, whereas PLAty-Crystals (PLACs) and Blue AGgregates (BAGs) either lack resolvable 26Mg-excesses or exhibit 26Mg deficits by up to ∼4‰. High precision, multiple collector SIMS analyses show that 6 of 7 SHIBs investigated fall on a single correlation line implying 26Al/ 27Al = (4.5 ± 0.2) × 10 −5 at the time of isotopic closure, consistent with the “canonical” 26Al abundance characteristic of internal isochrons in many calcium–aluminum-rich inclusions (CAIs). One SHIB sample exhibits Δ 26Mg ∗ consistent with a “supracanonical” 26Al/ 27Al ratio of (6.4 ± 0.5) × 10 −5. The PLAC hibonites contain highly anomalous titanium isotopic compositions, with δ 50Ti values ranging from −80‰ to almost +200‰, whereas SHIBs generally lack large Ti isotopic anomalies. Eight out of 11 26Al-free PLAC hibonite grains record 10B/ 11B excesses that correlate with Be/B; the inferred initial 10Be/ 9Be ratio of (5.1 ± 1.4) × 10 −4 is lower than the best-constrained 10Be/ 9Be of (8.8 ± 0.6) × 10 −4 in a CV CAI. The data demonstrate that 10Be cannot be used as a relative chronometer for these objects and that most of the 10Be observed in CAIs must be produced by irradiation of precursor solids in the early solar system. The lack of 26Al in PLAC hibonites indicates that significant amounts of 26Al were not formed in the same spallogenic processes that made 10Be in PLAC precursors. This is most easily understood as indicating very early formation of the PLAC hibonites, prior to the incorporation and mixing of 26Al into the solar nebula, although an alternative scenario, which invokes irradiation under different solar flare conditions, cannot be ruled out. Lithium isotopes are normal within uncertainties, probably reflecting contamination and/or postcrystallization exchange. The oxygen isotopic compositions of SHIBs and PLACs are all highly 16O-enriched, but are not derived from a homogeneous reservoir: Δ 17O values span a range of ∼−28‰ to −15‰. The ranges of 16O-enrichment in SHIBs and PLACs overlap and are less “anomalous” than the most 16O-enriched compositions found in meteorites [Kobayashi S., Imai H. and Yurimoto H. (2003) New extreme 16O-rich chondrule in the early solar system. Geochem. J. 37, 663–669]. Both PLACs and SHIBs formed in 16O-enriched reservoirs characterized by small-scale heterogeneities in the gas phase. If such heterogeneities were generated by an admixture of relatively 16O-poor gas created by self-shielding during CO photolysis and transported to the hot inner regions of the accretion disk, then this process must have been initiated very early on, prior to the arrival of fresh radioactivity into the inner solar system. Oxygen isotope heterogeneities persisted throughout the formation interval of PLACs, CAI precursors, and SHIBs which could be as long as 3 × 10 5 years based on 26Al records. One SHIB and one BAG exhibit mass fractionated oxygen isotopic compositions similar to those seen in FUN inclusions and in several platy hibonite crystals [Lee T., Mayeda T. K. and Clayton R. N. (1980) Oxygen isotopic anomalies in Allende inclusion HAL. Geophys. Res. Lett. 7, 493–496; Ireland T. R., Zinner E. K., Fahey A. J. and Esat T. M. (1992) Evidence for distillation in the formation of HAL and related hibonite inclusions. Geochim. Cosmochim. Acta 56, 2503–2520; Ushikubo T., Hiyagon H. and Sugiura N. (2007) A FUN-like hibonite inclusion with a large 26Mg-excess. Earth Planet. Sci. Lett. 254, 115–126]. The suite of mass-fractionated hibonites exhibit a range of isotopic properties, including 26Al/ 27Al ratios from below detection to a “canonical” level and oxygen and titanium anomalies that are not exceptional by PLAC standards. This suggests that F (fractionation) processes—evaporation under (oxidizing) conditions—are not necessarily associated with sampling a special isotopic reservoir.

Timescales for the evolution of oxygen isotope compositions in the solar nebula

We review two models for the origin of the calcium-, aluminum-rich inclusion (CAI) oxygen isotope mixing line in the solar nebula: (1) CO self-shielding, and (2) chemical mass-independent fractionation (MIF). We consider the timescales associated with formation of an isotopically anomalous water reservoir derived from CO self-shielding, and also the vertical and radial transport timescales of gas and solids in the nebula. The timescales for chemical MIF are very rapid. CO self-shielding models predict that the Sun has Δ 17O SMOW ∼ −20‰ (Clayton, 2002), and chemical mass-independent fractionation models predict Δ 17O SMOW ∼0‰. Preliminary Genesis results have been reported by McKeegan et al. (McKeegan K. D., Coath C. D., Heber, V., Jarzebinski G., Kallio A. P., Kunihiro T., Mao P. H. and Burnett D. S. (2008b) The oxygen isotopic composition of captured solar wind: first results from the Genesis. EOS Trans. AGU 89(53), Fall Meet. Suppl., P42A-07 (abstr)) and yield a Δ 17O SMOW of ∼ −25‰, consistent with a CO self-shielding scenario. Assuming that subsequent Genesis analyses support the preliminary results, it then remains to determine the relative contributions of CO self-shielding from the X-point, the surface of the solar nebula and the parent molecular cloud. The relative formation ages of chondritic components can be related to several timescales in the self-shielding theories. Most importantly the age difference of ∼1–3 My between CAIs and chondrules is consistent with radial transport from the outer solar nebula (>10 AU) to the meteorite-forming region, which supports both the nebular surface and parent cloud self-shielding scenarios. An elevated radiation field intensity is predicted by the surface shielding model, and yields substantial CO photolysis (∼50%) on timescales of 0.1–1 My. An elevated radiation field is also consistent with the parent cloud model. The elevated radiation intensities may indicate solar nebula birth in a medium to large cluster, and may be consistent with the injection of 60Fe from a nearby supernova and with the photoevaporative truncation of the solar nebula at KBO orbital distances (∼47 AU). CO self-shielding is operative at the X-point even when H 2 absorption is included, but it is not yet clear whether the self-shielding signature can be imparted to silicates. A simple analysis of diffusion times shows that oxygen isotope exchange between 16O-depleted nebular H 2O and chondrules during chondrule formation events is rapid (∼minutes), but is also expected to be rapid for most components of CAIs, with the exception of spinel. This is consistent with the observation that spinel grains are often the most 16O-rich component of CAIs, but is only broadly consistent with the greater degree of exchange in other CAI components. Preliminary disk model calculations of self-shielding by N 2 demonstrate that large δ 15N enrichments (∼ +800‰) are possible in HCN formed by reaction of N atoms with organic radicals (e.g., CH 2), which may account for 15N-rich hotspots observed in lithic clasts in some carbonaceous chondrites and which lends support to the CO self-shielding model for oxygen isotopes.

Photodissociation of Co−C Bond in Methyl- and Ethylcobalamin: An Insight from TD-DFT Calculations

The mechanism of Co-C bond photodissociation in methylcobalamin (MeCbl) and ethylcobalamin (EtCbl) has been examined by means of time-dependent density functional theory (TD-DFT). The present contribution extends our recent study (J. Phys. Chem. B 2007, 111, 2419-2422) where relevant excited states involved in the photolysis of MeCbl have been identified. To obtain reliable structural models, the high-resolution crystal structure of MeCbl was used as the source of initial coordinates. The full MeCbl was simplified by replacing the corrin side chains by H atoms and the resulting geometry was optimized. The model of EtCbl was generated from the simplified structure of MeCbl by replacing methyl group with ethyl. For both models, the low-lying singlet and triplet excited states have been computed along the Co-C coordinate at TD-DFT/BP86/6-31G(d) level of theory. These calculations reveal that the photodissociation process is mediated by the repulsive 3(sigmaCo-C-->sigma*Co-C) triplet state. The overall mechanism of photodissociation for both systems is similar but energetic details are different, reflecting the difference in Co-C bond strength in MeCbl and EtCbl. In both cases the key intermediate involved in Co-C bond photodissociation is identified as first excited state (S1). The S1 intermediate has mixed character: it can be described as predominantly dCo-->pi*corrin metal-to-ligand charge transfer (MLCT) state with contribution from sigma bond to corrin charge transfer (SBLCT) where upon electronic excitation the electron density shifts from the axial NIm-Co-C bonding to corrin ligand. The optimized geometry of the S1 indicates that the structure of the corrin remains essentially unchanged in comparison to ground state (S0). The major structural change occurs in the NIm-Co-C moiety, which becomes bent with elongated Co-C bond in S1 state. Finally, it is proposed that the photolysis of Co-C bond is in line with the mechanism of heme-CO photolysis, where participation of the dFe-->pi*porphyrin has been suggested.

Direct Observation of Ligand Dynamics in Cytochrome c

Horse heart cytochrome c (cyt c) has emerged as a paradigm for the study of protein folding, in large part because the covalently bound heme facilitates its characterization. The folding of reduced cyt c induced by photodissociation of CO from the CO-bound unfolded protein has been extensively studied. Following a nanosecond light pulse, four transitions with time constants of approximately 1-5, 50-100, 200-500, and 1000-10000 micros have been resolved. While originally thought to be associated with CO rebinding to two different partially folded states of cyt c, the two slower processes are now understood to reflect the bimolecular reassociation of CO followed by religation of His18, which by the base elimination mechanism is induced to dissociate after CO photolysis. Thus, it turns out that the two longer time constants do not report on protein folding but instead reflect the complexity of heme ligation. The two shorter time constants have been attributed to ligation at the heme center by Met65 or Met80 and His33 or His26 and have been used to estimate interchain diffusion rates of the protein. Here, to unambiguously determine the post-photodissociation steps involving CO, we have monitored the CO vibration following photodissociation with step-scan FT-IR spectroscopy. We have found that like the longer time scale processes, the 50-100 mus time scale process is associated not with protein dynamics but with CO ligand dynamics. The data clearly demonstrate that whatever the origins of the spectral changes, they clearly involve CO rebinding or changes in the environment of an already bound CO ligand. We speculate that the observed changes reflect His18 religation after fast geminate recombination of the CO. The data suggest that the associated time constant should not be used as a measure of interchain diffusion, and the results emphasize the importance of studying protein folding with probes that have inherently high structural resolution.