The formation of vibrationally excited heme upon photodissociation of carbonmonoxy myoglobin and its subsequent vibrational energy relaxation was monitored by picosecond anti-Stokes resonance Raman spectroscopy. The anti-Stokes intensity of the nu4 band showed immediate generation of vibrationally excited hemes and biphasic decay of the excited populations. The best fit to double exponentials gave time constants of 1.9 +/- 0.6 and 16 +/- 9 picoseconds for vibrational population decay and 3.0 +/- 1.0 and 25 +/- 14 picoseconds for temperature relaxation of the photolyzed heme when a Boltzmann distribution was assumed. The decay of the nu4 anti-Stokes intensity was accompanied by narrowing and frequency upshift of the Stokes counterpart. This direct monitoring of the cooling dynamics of the heme cofactor within the globin matrix allows the characterization of the vibrational energy flow through the protein moiety and to the water bath.

A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59°C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-'.The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native -unfolded) states. The signal at 1633 cm-1, corresponding to the fl-sheet structure, achieved 15 ± 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-jump. The disruption of fl-sheet therefore appears to be subject to a delay of -1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions.Questions concerning the physical and chemical nature of protein folding are among the most challenging in biological research (1-4). Folding and unfolding events have seldom been studied on time scales shorter than milliseconds. Internal motions of macromolecules such as rotations about single bonds, chemical exchange reactions, diffusion over molecular dimensions, and barrier crossing processes can occur on nanosecond or even picosecond time scales, so protein structure reorganization might be expected to involve ultrafast intermediate steps. An example is the recent report of tens-ofmicroseconds folding in cytochrome c (5). Some of the faster processes in protein folding might involve relatively small alterations in electronic structure. Therefore the probes used to examine them must be sensitive to subtle changes in, for example, nonbonded interactions, weaker chemical bonds, charge distributions, and motions of pieces of the structure. For this reason we decided to use transient infrared (IR) spectroscopy (6-8), which is structure sensitive at a chemicalbond resolution, to identify any ultrafast folding steps.The IR spectra of proteins in the region of the amide vibrations of the polypeptide structures are well known to be sensitive to the state of the protein. For example, there are distinct differences between the IR spectra of random coil, a-helical, }3-sheet, 13'-sheet, and turn structures of polypeptides (9). These differences arise from the dependence of interactions between the various amide groups on the local polypeptide structures. One can therefore conceive of carrying out time-resolved IR capable of following the kinetics of structure change as it affects these different spatial regions of the polypeptide backbone. Experiments on the kinetics of folding also require that the system be triggered to suddenly change. For this purpose we have developed an ultrafast temperature jump...

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Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that was completed within the instrument response time. The intensity decrease was followed by an intensity recovery with a time constant of Ϸ50 ps and lasted up to 1 ns. The time-resolved UVRR study of the myoglobin mutants demonstrated that the hydrophobicity of environments around Trp-14 decreased, whereas that around Trp-7 barely changed in the primary protein response. The present data indicate that displacement of the E helix toward the heme occurs within the instrument response time and that movement of the FG corner takes place with a time constant of 2 ps. The finding that the instantaneous motion of the E helix strongly suggests a mechanism in which protein structural changes are propagated from the heme to the A helix through the E helix motion.hemeprotein ͉ protein dynamics ͉ resonance Raman spectroscopy ͉ time-resolved spectroscopy P roteins are endowed with both stiff and flexible properties; hence, their dynamics are closely associated with structure and function. Because allosteric proteins, in general, propagate conformational changes over considerable distances, how these conformational changes are generated and transmitted is of major interest for understanding the regulatory, kinetic, and recognition properties of proteins (1-3). A variety of experimental evidence suggests that rapid and long-range propagation of conformational changes through the core of protein plays a vital role in allosteric communication. For example, the cooperative oxygen-binding properties of hemoglobin (Hb) result from a change in quaternary structure, which is initiated by ligand binding/release at the heme (ligand binding site). Therefore, if the pathway by which one quaternary structure is converted to the other quaternary structure is structurally characterized, our understanding how a protein performs its function will be greatly advanced. The ligand-induced dynamics of myoglobin (Mb) are a basic subject for studying such features in proteins. Although Mb is a monomeric protein, the threedimensional structure of Mb is closely similar to that of a subunit of Hb. Thus, the structural changes of Mb can be regarded as a model for the tertiary structural events that cause the quaternary structural change of Hb.

The heme environments of Met 95 and His 77 mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band ( Fe-His ) at 214 cm ؊1 , and the Fe-CO versus CO plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the Fe-His band but gave a Fe-Im band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a fivecoordinate heme complex. Taken Heme-containing signal-transducing proteins (1-3) respond to diatomic molecules, which act as physiological, environmental messengers. This has attracted the attention of biophysical chemists. The O 2 sensing proteins so far identified include FixL (an oxygen-sensing kinase of Rhizobia meliloti) (1, 4), HemAT (an oxygen sensor heme protein discovered from Bacillus subtilis (HemAT-Bs) and Halobacterium salinarium (HemAT-Hs)) (5, 6), PDEA1 (7), and putatively a heme protein from E. coli (designated Escherichia coli DOS) (8). There is only one CO sensor protein known (CooA, a CO-binding transcriptional regulation factor from Rhodospirillum rubrum) (9, 10) and one NO sensor (soluble guanylate cyclase) (11,12). In each case, binding of an external ligand to the heme located in an N-terminal sensory domain transmits a signal to the functional C-terminal domain (either enzymatic or DNA binding). We are curious to know how these proteins recognize a specific diatomic molecule to generate the appropriate physiological response and what kind of structural changes occur to transmit the signal from the sensory domain to the functional domain.The sensory domain of FixL belongs to the large family of signal-transducing PAS domain 1 proteins, whereas those of HemAT, CooA, and soluble guanylate cyclase do not. The PAS domain proteins found in eukarya, archaea, and bacteria contain a partly conserved tertiary structure despite their limited sequence homology (Ͻ15%) and dissimilar cofactors (13). Although structures of three PAS proteins including the human voltage sensor (HERG) (14), the rhizobial oxygen sensor (FixL) (15, 16), and bacterial light sensor (PYP) (17) have been solved, interactions between the sensory domain and the functional domain are not clearly understood. Namely, hydrophobic interactions seem important to regulate the K ϩ channel of HERG, whereas polar interactions in the EF loop of the PAS domain seem to be essential to PYP. In the case of FixL, either a protein conformational change associated with the location of the heme iron (in-pla...

The development of methods for the highly selective and efficient conversion of abundant organic resources into valuable products is crucial for a sustainable society. To achieve this goal, extensive studies on the methodology of efficient material conversion with metal complexes as catalysts have been made for a long time. [1,2] High-valent metal-oxo species are key intermediates in biological oxidations by metalloenzymes (mainly heme and non-heme iron enzymes), which catalyze the oxygenation of hydrocarbons in metabolic and catabolic processes. [3,4] These oxygenases involve high-valent metal-oxo species as reactive species that arise by reductive activation of molecular oxygen coupled with proton transfer. [5][6][7] Peroxides such as hydrogen peroxide can lead to a so-called "peroxide shunt" to perform the catalytic oxygenation; this mechanism is found for cytochrome P450 [8] and methane monooxygenase. [9] Thus, a number of model systems for these enzymatic oxidations have been developed to elucidate the reaction mechanisms and to perform effective catalytic oxygenation of external substrates with metal complexes involving the formation of high-valent metal-oxo species.[10-12] These systems usually require organic solvents and excess amount of organic or inorganic peroxides as both oxidants and oxygen sources. Moreover, in such cases, the reaction pathways become complicated and give multiple products. Consequently it is difficult to control the product distribution that arises mainly from the inevitably produced radical species. [13,14] Another strategy to generate a high-valent metal-oxo species has been recognized in the oxygen-evolving complex (OEC) in Photosystem II (PS II) for the photosynthesis to oxidize water to produce dioxygen.[15] At the OEC, a manganese(V)-oxo species has been proposed to be formed by proton-coupled electron transfer (PCET), and the deprotonation of coordinated water and the oxidation of the metal center are thought to occur concertedly. [16] This strategy has been applied to form and isolate high-valent metal-oxo species to perform stoichiometric oxidation reactions; [17] however, it has not been applied to catalytic oxidations with transition-metal complexes as catalysts in water.Inspired by the reactions at the OEC in photosynthesis, we have tried to establish a novel catalytic oxygenation system using water as both the solvent and the oxygen source by virtue of PCET. [18,19] We report herein the formation of a novel ruthenium(IV)-oxo complex and its reactivity toward highly efficient and selective catalytic oxygenation and oxidation reactions of various hydrocarbons in water, which can be used as an oxygen source.We synthesized a novel bis-aqua Ru II complex, [Ru II -(tpa)(H 2 O) 2 ](PF 6 ) 2 (1; tpa = tris(2-pyridylmethyl)amine) (Figure 1 a, b), by the treatment of [Ru II Cl(tpa)] 2 (PF 6 ) 2 [20] with AgPF 6 in water. Complex 1 exhibits a reversible twostep deprotonation-protonation equilibrium, and the two pK a values were determined by UV/Vis spectroscopic titration (see F...

Studies on the structural relaxation of myoglobin following CO photolysis revealed that the structural change of heme itself caused by the cleavage of the Fe−CO bond is completed within the instrumental response time (∼2 ps) of the time-resolved resonance Raman apparatus used. In contrast, changes in the intensity and frequency of the iron-histidine stretching [ν(Fe−His)] mode were found to occur in the picosecond regime. The ν(Fe−His) band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous in contrast with the changes observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. Same behaviors were observed for the model compound of the heme part without protein matrix. Therefore the intensity change in ν(Fe−His) is not associated with protein relaxation following the CO photodissociation. The band position of the ν(Fe−His) mode changed with a time constant of about 100 ps, whereas that of the model compound without the protein matrix showed no shift. This indicates that tertiary structural changes of the protein occurred in a 100 ps range.