Cytochrome pulse radiolysis

EJ. Land and A.J. Swallow, One-electron reactions in biochemical systems as studied by pulse radiolysis. V. Cytochrome c. Arch. Biochem. Biophys. 145, 365-372 (1971). 

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. 

E. Mechanistic Studies on P. pantotrophus Cytochrome cdi 1. Electron Transfer Studied by Pulse Radiolysis... 

The spectrum in the Soret region, indicative of the c-type center of the enzyme, immediately following the reduction is not identical to that obtained when the enzyme is fully reduced under steady-state/equilibrium conditions. This suggests that the reduced c-type cytochrome center formed under the conditions of the pulse radiolysis experiment retained the His/His coordination. 

Other examples using pulse radiolysis include (a) studies on cytochrome c-di nitrate reductase from Thio-sphaera pantotropha to provide evidence for a fast intramolecular electron transfer from c-heme to rate constant for the reaction of... 

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. 

Electron transfer between the iron centre in cytochrome c and a transition metal bound at the periphery of the protein has been studied in cases where Cu" 677 and the Rum(NH3)5 group678 are bound to His-33. In the latter case, the Ruu-cytochrome c(III) form can be generated by pulse radiolysis. The intramolecular transfer of an electron between Ru" and Fe111 can be measured. This presents a useful model for electron transfer from the iron via the heme group and the protein. 

The reaction Fe ccp/Fe cytc + Fe ccp/Fe cytc proceeds with AE s 0.4V. The reaction h j been moj ored both by pulse radiolysis, and by simple mixing of Fe ccp + Fe1 cytc, with equivalent results k 0.25 0.07 s (figure 10) It is interesting that a dependence of rate on the primary structure of the protein is observed (at constant AG) for horse cy1j.c/ccp(yeast) k = 0.25 s but for yeast. cytc/(yeast) ccp k = 4 s 1 and for tuna cytc/yeast ccp k s 0.1 s, even though the general three dimensional structures are essentially identical for horse, tuna and yeast cytochromes c. These determinations disprove an earlier suggestion based on modulated excitation spectroscopy, that k - 10 s. Clearly the rate is slow,... 

Many 1-alkyl-l-hydropyridinyl radicals are not persistent in aqueous medium. The bimolecular decay reaction has been investigated for 66 and 70 and a mechanism consistent with products and kinetics advanced.239 The reactions of 70, its 3-carboxamide isomer, and the pyridinyl radical derived from nicotinamide adenine dinucleotide (NAD) with cytochrome c have been investigated by pulse radiolysis and rates established.240... 

Kobayashi, K., Koppenh fer, A., Ferguson, S. J., and Tagawa, S., 1997, Pulse radiolysis studies on cytochrome cd nitrite reductase from Thiosphaera pantotropha Evidence for a fast intramolecular electron transfer from c heme to d, heme, RiocAemtstry 36 1361 In 13616. 

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. 

Some of the first protein systems where pulse radiolysis was used to help determine mechanism were those of blue copper proteins. These are proteins that are blue in solution and contain what are known as type (I) and type (2) copper centers. Two of the most well-known and well-characterized examples of these are azurin and cytochrome c. It was the studies of these systems that opened up the field of long-distance electron transfer in proteins and, by using the protein structure as a framework for electron transfer through space and through bonds, allowed for the development of a broad theoretical basis and many fascinating experiments on long-range electron transfer. Here, I will limit the discussion to electron transfer studies in azurin as illuminated by pulse radiolysis studies. ... 

Even if pulse radiolysis has been used [13], most studies of the initial electron entry into cytochrome oxidase are based on the finding that at low ionic strength cytochrome c forms a strong electrostatic complex with the oxidase [14]. Cytochrome c itself cannot be photoactivated, but in the complex with the enzyme it can be reduced by photochemically generated radicals, such as that of 5-diazariboflavin [15]. Another approach has been to employ a photoinduced uroporphyrin/NADH reduction system [16]. 

In 1993 Weiss, Riley, and co-workers reported a study on purported SOD mimics by stopped-flow UV-vis spectroscopy (428) in which they assessed reactivity by following the decay of the superoxide absorption at 245 nm. Two of the earlier techniques that had been used to assess SOD activity included observation by UV-vis spectroscopy of the oxidation of nitroblue tetrazolium (NBT) (68) or the oxidation of a cytochrome c by superoxide (52). Both systems used superoxide from an in situ generator, frequently xanthine oxidase, wherein the complex being analyzed was compared to a calibrated oxidation of the chromophore alone and in the presence of MnSOD. The direct observation of the decrease in the superoxide signal with time by UV-vis is also possible, and superoxide may be introduced as a solution (428) or generated, in some cases, by pulsed radiolysis (79, 80). In these direct observation experiments, the rate of decay of superoxide in the presence of the complex is compared to the rates of decay of superoxide alone and in the presence of one unit of activity of MnSOD. In all cases, the systems are usually referenced, or calibrated, against the same set of conditions with MnSOD. Due to interactions with cytochrome c with components of assay mixtures other than superoxide, false readings of activity were often observed for some early SOD mimics. The NBT, stopped-flow, or pulsed radiolysis techniques have tended to provide the more accurate answers on the ability of reputed MnSOD mimics. To be considered active in any manner with respect to the decay of superoxide in the stopped-flow analyses, Weiss et al. stated that compounds based on their analyses needed to exhibit kcat values in excess of 10B 5 M 1 s 1 (428). 

Vibrational spectroscopy of the electronically excited state pulse radiolysis/time-resolved resonance Raman study of the triplet y3-carotene. J Am Chem Soc 101 1355-1357 De Paula JC, Ghanotakis DF, Bowlby NR, Dekker JP, Yocum CF and Babcock GT (1990) Chlorophyll-protein interactions in Photosystem II. Resonance Raman spectroscopy of the D1 D2-cytochrome b559 complex and the 47 kDa protein. In Baltscheffsky M (ed) Current Research in Photosynthesis, pp 643-646, Kluwer Academic Publishers, Dordrecht Frank HA and Cogdell RJ (1993) Photochemistry and functions of carotenoids in Photosynthesis. In Young A and Britton G (eds) Carotenoids in Photosynthesis, pp 252-326. Chapman Hall, London... 

Application of pulse-radiolysis techniques (see Sect. 1.4 for more details on the experimental set-up) revealed that the following intramolecular (Eqs 1.6 and 1.7) and intermolecular (Eq. 1.8) electron-transfer reactions, where cyt c represents cytochrome c, all exhibit a significant acceleration with increasing pressure. The reported volumes of activation are —17.7 0.9, -18.3 0.7, and —15.6 0.6 cm mol respectively, and clearly demonstrate a significant volume collapse upon reaching the transition state [63]. 

The potential of pulse radiolysis for studying biological redox processes, particularly of macromolecules, was recognized rather early. It was initially employed for investigating radiation-induced damage and, later on, as an effective tool for resolving electron transfer processes to and within proteins. Cytochrome c, a well-characterized electron-mediating protein, was the first to be... 

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