Myoglobin photolysis

V. Srajer, T. Teng, T. Ursby, C. Pradervand, Z. Ren, S. Adachi, W. Schildkamp, D. Bourgeois, M. WuRf, and K. Moffat, Photolysis of the carbon monoxide complex of myoglobin nanosecond time-resolved crystallography. Science 274, 1726-1729 (1996). 

The kinetics of reactions of NO with ferri- and ferro-heme proteins and models under ambient conditions have been studied by time-resolved spectroscopic techniques. Representative results are summarized in Table I (22-28). Equilibrium constants determined for the formation of nitrosyl complexes of met-myoglobin (metMb), ferri-cytochrome-c (Cyt111) and catalase (Cat) are in reasonable agreement when measured both by flash photolysis techniques (K= konlkQff) and by spectroscopic titration in aqueous media (22). Table I summarizes the several orders of magnitude range of kon and kQs values obtained for ferri- and ferro-heme proteins. Many k0f[ values were too small to determine by flash photolysis methods and were determined by other means. The small values of kQ result in very large equilibrium constants K for the... 

It is quite evident that the ferrous complexes of porphyrins, both natural and synthetic, have extremely high affinities towards NO. A series of iron (II) porphyrin nitrosyls have been synthesized and their structural data [11, 27] revealed non-axial symmetry and the bent form of the Fe-N=0 moiety [112-116]. It has been found that the structure of the Fe-N-O unit in model porphyrin complexes is different from those observed in heme proteins [117]. The heme prosthetic group is chemically very similar, hence the conformational diversity was thought to arise from the steric and electronic interaction of NO with the protein residue. In order to resolve this issue femtosecond infrared polarization spectroscopy was used [118]. The results also provided evidence for the first time that a significant fraction (35%) of NO recombines with the heme-Fe(II) within the first 5 ps after the photolysis, making myoglobin an efficient N O scavenger. 

The kinetics of myoglobin oxidation and reduction have been studied by a variety of experimental techniques that include stopped-flow kinetics, pulse radiolysis, and flash photolysis. In considering this work, attention is directed first at studies of the wild-type protein and then at experiments involving variants of Mb. 

An alternative application of flash photolysis to study myoglobin electron transfer kinetics has been employed by Hofifinan and co-workers 156). In this approach, the photoactive zinc-substituted derivative of Mb is mixed with an equivalent amoimt of ferricytochrome bs to form an electrostatically stabilized binary complex. Upon transient irradiation, the strongly reducing Zn-Mb intermediate is formed, and the kinetics of ferricytochrome reduction within the preformed complex can be monitored spectrophotometrically. The resulting kinetics represents a mixed-order process consistent with electron transfer both within the electrostatically stabilized complex and between the dissociated components of the complex. 

Fig. 7. The instrumental set up at XI1 /DORIS for time resolved data collection with a linear detector for CO myoglobin following laser photolysis of the ligand. A section of the diffraction pattern with stationary crystal, stationary detector is recorded with a linear detector and (b) shows the time course of three reflections before and after the laser flash (from Bartunik et al. 1982)...

Srajer, V., Teng, T. Y, Ursby, T., Pradervand, C., Ren,Z., Adachi, S., Schildkamp, W., Bourgeois, D., Wulff, M., and Moffatt, K. 1996. Photolysis of the carbon monoxide complex of myoglobin Nanosecond time-resolved crystallography. Science 274 1726-29. 

Teng, T. Y, Srajer, V., and Moffat, K. 1994. Photolysis-induced structural changes in single crystals of carboiunonoxy myoglobin at 40 K. Struct. Biol. 1 701-5. 

Fe(CO)5], 14, and [Fe2(CO)9], 15, along with other metal carbonyls have been reported in a patent as being applied as a transdermal patch or cream [190]. Subsequently, [Fe(CO)5] was shown to release CO to myoglobin on photolysis [58, 59]. 

Cell viability studies of HT29 colon cancer cells treated with the CO-releasing compound [Mn(CO)3(tpm)]PF6, 33 R = H, revealed a significant photoinduced cytotoxicity comparable to that of established agent 5-fluorouracil [205]. Subsequently, [Mn(CO)3(tpm)]PF6, 33, was modified by replacing R with peptides to permit cellular recognition [206]. 34, R = H, Me, releases nearly 2 mol of CO to myoglobin on photolysis while 35, E = lone pair, O, S, releases approximately 1 mol of CO. The n-octanol/water partition coefficients were also measured [207]. 

The picosecond internal dynamics of myoglobin was explored by measuring inelastic neutron scattering by Smith et al. [25]. At low temperatures they found the dynamics to be harmonic while at higher temperatures a considerable quasielastic scattering was detected. Agreement between the experimentally observed spectra and that calculated from molecular dynamics simulations also showed evidence for restriction of the conformational space sampled at 80 K relative to 300 K. On the basis of these results it was concluded that the protein is trapped in local minima at low temperatures in accord with the multiple substate model suggested by low temperature flash photolysis experiments and previous molecular dynamics simulations. Comparison of atomic fluctuation data sets collected at both 325 K and 80 K confirms that the room temperature... 

Fraunfelder and co-workers [65-68] have found the same relationship in the reduction kinetics of CO and O2 ligand coordination bonds with the complex-forming Fe ions of the heme group myoglobin upon photodissociation of these bonds induced by laser photolysis of 10 s duration. The formation of Fe-CO and Fe-02 bonds after photolysis was registered with a spectrophotometer by restoration of the mother compound absorption spectrum in the wide range of times (10 -10 s) and temperatures (2-300 K). Before photolysis the six-coordinate Fe ion is in the heme plane. The coordination bond break causes not only the ligand shift but also... 

A variety of additional measurements have been made on the photolysis behavior of myoglobin and the related protein, hemoglobin. These include room-temperature transient electronic absorption studies in the nanosecond range,521 nanosecond-to-picosecond resonance Raman spectroscopy,522,523 and low-temperature transient electronic absorption measurements in regions other than the Soret band.524... 

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