Flash photolysis myoglobins

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... 

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. 

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... 

Slower processes occurring in the /as time scale were also studied by flash photolysis. Interpretation of the kinetic data requires a four-state model shown in equation (4) where MbL represents myoglobin with the small ligand bound at six-coordinated Fe(II), Mb L (generated by photodissociation) represents the short-lived... 

Table 8.2. Rate Constants Following Flash Photolysis of Myoglobin-XY Complexes Analyzed According to Scheme 8.6...

Figure 2.4 Time-dependence of rate constant in the initial stage of a fast reaction. Plots of reciprocal change of absorbance (proportional to Ac ) against square root of time, for the recombination after flash photolysis of CO and O2 with myoglobin and haemoglobin in glycerol-water mixtures at -80 C. From Ref. [ 15,b).

Laser flash photolysis is often used to measure the binding rate of CO to heme proteins, such as myoglobin (Mb), because CO dissociates from the bound state relatively easily on absorption of energy from an intense and short pulse of light. The reaction is usually run under pseudo-first-order conditions. For a reaction in which [Mb]o = 10 mmol dm" , [CO] = 400 mmol dm" , and the rate constant is 5.8 X 10 dm mol" s" , plot a curve of [Mb] against time. The observed reaction is Mb + CO —> MbCO. 

The use of well collimated and high intensity synchrotron radiation beams is essential to reach the necessary time intervals without losing the statistical significance in the observed diffracted intensities. The white beam Laue technique has already been proven to facilitate studies down to the picosecond time regime for studies such as recombination of CO in myoglobin after flash photolysis. Nanosecond resolution has been obtained in a study of laserannealing of defects in a silicon crystal. 

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)...

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