Flash photolysis cytochrome

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... 

Recent advances in measuring the kinetics of the various electron-transfer steps in this system have been achieved by use of flash photolysis of ruthenated derivatives of cytochrome c (Ru-Cc) (17-19). In these studies [Ru(bpy)3]2+ is covalently bound to a surface residue at a site that does not interfere with the docking of cytochrome c to cytochrome c oxidase. Solutions are then prepared containing both Ru-Cc and cytochrome c oxidase, and the two proteins associate to form a 1 1 complex. Flash photolysis of the solution leads directly to the excitation of the RuII(bpy)3 site, which then reduces heme c very rapidly. This method thus provides a convenient means to observe the subsequent intracomplex electron transfer from heme c to cytochrome c oxidase and further stages in the process. 

The systems that we investigated in collaboration with others involved intermolecular and intramolecular electron-transfer reactions between ruthenium complexes and cytochrome c. We also studied a series of intermolecular reactions between chelated cobalt complexes and cytochrome c. A variety of high-pressure experimental techniques, including stopped-flow, flash-photolysis, pulse-radiolysis, and voltammetry, were employed in these investigations. As the following presentation shows, a remarkably good agreement was found between the volume data obtained with the aid of these different techniques, which clearly demonstrates the complementarity of these methods for the study of electron-transfer processes. 

Bhattacharyya, A.K., Lipka, J.J., Waskell, L. and Tollin, G. (1991) Laser flash photolysis studies of the reduction kinetics of NADPH cytochrome P-450 reductase. Biochemistry, 30 (3), 759-765. 

Intramolecular electron transfer from Ru(II) to Fe(III) in (NH3)3Ru(II) (His-33)cyt(Fe(III)) induced by pulse-radiolysis reduction of Ru(III) in the (NH3)5Ru(III) (His-33)cyt(Fe(III)) complex were investigated [84]. The results obtained differ from those of refs. 77-80 where flash photolysis was used to study the similar electron transfer reaction. It was found [84] that, over the temperature range 276-317 K the rate of electron transfer from Ru(II) to Fe(III) is weakly temperature dependent with EA 3.3 kcal mol 1. At 298 K the value of kt = 53 2 s"1. The small differences in the temperature dependence of the electron tunneling rate in ruthenium-modified cytochrome c reported in refs. 77-80 and 84 was explained [84] by the different experimental conditions used in these two studies. 

Furukawa Y, Ishimori K, Morishima I. Oxidation-state-dependent protein docking between cytochrome c and cytochrome bs high-pressure laser flash photolysis study. Biochemistry 2002 41 9824-32. 

SjogrenT, Svensson-Ek M, Hajdu J, Brzezinski P. Proton-couple structural changes upon binding of carbon monoxide to cytochrome cdl A combined flash photolysis and X-ray crystallography study. Biochemistry 2000 39 10967-74. 

One advantage of Raman spectroscopy is that it is relatively easy to perform time-resolved spectra on a sub-millisecond time scale. Therefore it is possible to obtain spectra of a reaction intermediate that decays rapidly. For example, coupling resonance Raman to flash photolysis resulted in the detection of the much-hypothesised ferryl intermediate in cytochrome c oxidase [207]. 

CO reacts with Fef+ to yield a characteristic absorption maximum near 590 nm. The CO-bound form is photosensitive. Thus, the flash-photolysis technique has been involved in most of the kinetic investigations of the reaction of cytochrome c oxidase with O2, as described below. 

Evidence for more complex ET processes came from studies in which photo chemically generated reductants injected electrons into preformed Fe-cytochrome b lYt-cytochrome c complexes. In one study, the rate of c ET (1.7 X 10 s ) was reported to depend on viscosity and surface mutations. A later laser-flash photolysis study found a rate-limiting second-order reduction of Fe-cytochrome i s/Fe-cytochrome c complexes and no sign of satmation, suggesting that the intracomplex ET rate was greater than lO s-. ... 

Pulse Radiolysis A technique related to flash photolysis pulse radiolysis uses very short (nanosecond) intense pulses of ionizing radiation to generate transient high concentrations of reactive species. See Salmon, G. A. and Sykes, A.G., Pulse radiolysis, Methods Enzymol. 227, 522-534, 1993 Maleknia, S.D., Kieselar, J.G., and Downard, K.M., Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry. Rapid Commun. Mass Spectrom. 16, 53-61, 2002 Nakuna, B.N., Sun, G., and Anderson, V.E., Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis, Free Radic. Biol. Med. 37, 1203-1213, 2004 BataiUe, C., Baldacchino, G., Cosson, R.P. et al., Effect of pressure on pulse radiolysis reduction of proteins, Biochim. Biophys. Acta 1724, 432-439, 2005. 

A combination of stopped-flow mixing and laser-flash photolysis of the newly generated species is finding increasing use in studies of biological reactions. In a recent example [16], a ferrocytochrome a3-CO adduct was produced by the stopped-flow mixing of the fully reduced cytochrome caaj and CO. The follow-up photolysis caused rapid dissociation of CO. The recombination of the reduced ferrocytochrome and CO, and the reaction with O2 were then observed as kinetic steps. 

Incubation of oxidized cytochrome oxidase under an atmosphere of CO results in the formation of the so-called mixed-valence state, a form in which the cytochrome a3-CuB site is reduced and has bound CO, whereas cytoehrome a and Cua remain oxidized [22]. The dissociation of the bound CO by flash photolysis in the absence of O2 induces an electron backflow from the reduced cytochrome 03-Cub site to the oxidized redox centers, as first discovered by Boelens et al. [23] and later confirmed by Brzezinski and Malmstrom [24]. These authors thought that the electron acceptor is Cua, but later it has been shown [25] that both cytochrome a and Cua are sequentially reduced (see Section 3.4.2). In addition, it was found that oxidation of cytochrome 03 causes deprotonation of an acid-base group in its vicinity [26], as will be discussed further in Section 3.4.4. 

Flash photolysis of the mixed-valence cytochrome oxidase-CO compound leads to a drop in the apparent reduction potential of cytochrome ay, and this results in a backflow of electrons from cytochrome a to the other redox sites. Three kinetic phases can be observed [26, 41], with relaxation rate constants (i.e., the sum of the rate constants for the forward and reverse reactions of the equilibria involved) of 3 X 10, 2 X 10, and 10 s . The first two phases involve ET fl3 —> a and a Cua, respectively the third phase is strongly pH-dependent and represents further electron re-equilibration between a-i and a, following the dissociation of a proton caused by the initial oxidation of a-i [26, 41a], as will be further discussed in Section 3.4.4. Direct electron equilibration between ai and Cua is not observed. 

Recently we carried out kinetic studies with Hildenborough and Miyazaki cytochrome c3 using deazariboflavin semiquinone (dRf ), MV +, and propylene diquat (PDQ +), produced by laser flash photolysis, as reductants (37). Initially, all three reactions were accurately second order, consistent with all hemes being reduced with the same rate constant or with a single site reduced, followed by fast intramolecular electron transfer to reduce the remaining three hemes. However, by measuring reduction kinetics with cytochrome c3 poised at different extents of reduction, the kinetics of reduction of individual hemes could be resolved. Thus, reduction of cytochrome c3 in approximately 5% steps and application of the known macroscopic redox potentials (see previous section) enabled calculation of the concentration of each heme (c.) at each stage of reduction. The plot of kohs versus percent reduction can thereby be fitted to solve for the rate constant for each heme (kt) ... 

H. Leong, A. Markowitz et al. (2000). Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates A CO flash photolysis study. Biochemistry 39, 5731-5737. 

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