DNA-mediated electron transfer from a modified base to ethidium: pi-stacking as modulator of reactivity

BACKGROUND: The DNA double helix is composed of an array of aromatic heterocyclic base pairs and, as a molecular pi-stack, represents a novel system for studying long-range electron transfer. Because many base damage and repair processes result from electron-transfer reactions, the ability of DNA to mediate charge transport holds important biological implications. Seemingly contradictory conclusions have been drawn about electron transfer in DNA from the many different studies that have been carried out. These studies must be reconciled so that this phenomenon can be understood both at a fundamental level and in the context of biological systems. RESULTS: The photoinduced oxidation of a modified base, 7-deazaguanine, has been examined as a function of distance, sequence, and base stacking in DNA duplexes covalently modified with ethidium. Over ethidium/deazaguanine separations of 6-27 A, the photooxidation reaction proceeded on a subnanosecond time scale, and the quenching yield exhibited a shallow distance dependence. The efficiency of the reaction was highly sensitive to small changes in base composition. Moreover, the overall distance-dependence of the reaction is sensitive to sequence, despite the constancy of photoexcited ethidium as acceptor. CONCLUSIONS: The remarkable efficiency of deazaguanine photooxidation by intercalated ethidium over long distances provides new evidence for fast electron-transfer pathways through DNA. By varying sequence as well as reactant separation, this work provides the first experimental demonstration of the importance of reactant stacking in the modulation of long-range DNA mediated electron transfer.     

Oxidative charge transfer To repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metallointercalators

Potent oxidants which intercalate in DNA serve as tools to probe DNA-mediated electron-transfer reactions. A photoexcited rhodium intercalator, Rh(phi)2DMB3+ (phi = 9,10-phenanthrenequinone diimine and DMB = 4,4'-dimethyl-2,2'-bipyridine), tethered to DNA, promotes both oxidative damage to 5'-GG-3' doublets in DNA and the repair of thymine dimers from a remote site on the DNA duplex. DNA-mediated repair of a thymine dimer lesion by charge transfer from the tethered rhodium intercalator is quantitative, albeit with low photoefficiency, occurs in an intraduplex reaction over long range (36 A), and requires that the intervening bases be paired. When both oxidative reactions, repair and oxidative damage, are monitored on the same duplex, competition is evident; the presence of both a 5'-GG-3' site and the thymine dimer diminished the dimer repair efficiency by 20-40% and decreased damage at the 5'-GG-3' sites 2-fold compared to similar sequences lacking either the guanine doublet or thymine dimer, respectively. In addition to damage at the 5'-G of 5'-GG-3' sites, we also observe oxidation at the 3'-G of the 5'-GT<>TG-3' tetrad only in the presence of thymine dimer. Overall, the yield of repaired thymine strand was at least 10 times higher than the yield of oxidized guanine in the same sequences. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Dipyridophenanzine (dppz) complexes of ruthenium(III), less potent oxidants which intercalate in DNA, oxidize 5'-GG-3' doublets efficiently but cannot trigger the repair of the thymine dimer lesion. Oxidative damage to DNA from a distance, mediated by the DNA base pair stack, can, however, be utilized to probe the disruption in the base stack generated by the thymine dimer. The presence of the dimer does not diminish oxidation by a Ru(III) intercalator at a distal guanine doublet, suggesting that the disruption caused by the dimer does not block charge transfer through the DNA duplex. DNA-mediated electron-transfer reactions of metallointercalators therefore serve to illustrate important aspects of radical migration and its consequence with respect to reactions at a distance through the DNA base pair stack.     

Mechanisms of DNA damage by chromium(V) carcinogens

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.     

Effects of secondary structure on DNA and RNA cleavage by diplatinum(II)

The photochemistry of Pt2(pop)44- with nucleic acids has been studied using radiolabeled oligomers of DNA and RNA and high-resolution electrophoresis (pop is P2O5H22-). Photolysis of Pt2(pop)44- with duplex DNA produces an even cleavage ladder and relatively little enhancement of cleavage upon treatment with piperidine. In contrast, the cleavage pattern is far less regular with single-stranded DNA, and there is a large enhancement in cleavage upon treatment with piperidine. Accordingly, photolysis of Pt2(pop)44- with the DNA hairpin 5'-d[ATCCTATTTATAGGAT] produces a much larger piperidine enhancement at the loop and end nucleotides than in the stem. There is an additional piperidine enhancement that occurs selectively at guanine residues either in RNA or in DNA at low Mg2+ concentrations that is attributed to outer-sphere electron transfer on the basis of the known excited-state redox potentials of Pt2(pop)44- and the expected oxidative chemistry of guanine. The extent of guanine oxidation is higher compared to the extent of sugar oxidation at low Mg2+ concentrations, which can be attributed to a shallower distance dependence for electron transfer compared to that for atom transfer. The effects of Mg2+ and piperidine or aniline treatment were examined on stem-loop structures of DNA and RNA and gave partial images of the expected secondary structures.     

Mechanistic studies on the neighboring base damage induced by KMnO4 oxidation of 8-oxoguanine in DNA

New types of DNA substrates containing an 8-oxoguanine residue (8-oxo-G) were prepared in order to examine the mechanisms for the neighboring base damage initiated by KMnO4 oxidation of the 8-oxo-G. The results obtained from the reactions suggested that the damage at remote sites in the single strands can be explained by an electronic interaction (redox reaction) between an oxidized 8-oxo-G species and the base (to be damaged), which are close each other in a loop structure. For the inefficient damage observed in duplex substrates, electron transfer through stacked bases might be involved.     

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G.     

Alpha-tocopherol induces oxidative damage to DNA in the presence of copper(II) ions

There is currently much interest in the possibility that dietary antioxidants may confer protection from certain diseases, such as atherosclerosis and cancer. The importance of alpha-tocopherol (vitamin E) as a biological antioxidant is widely recognized. However, pro-oxidant properties of alpha-tocopherol have been observed in chemical systems, and it has been reported that the vitamin can induce tumor formation and act as a complete tumor promotor in laboratory animals. In the present communication, we find that alpha-tocopherol can act as a potent DNA-damaging agent in the presence of copper(II) ions, using a simplified, in vitro model. alpha-Tocopherol was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base oxidation and backbone cleavage. Neither alpha-tocopherol nor Cu(II) alone induced DNA damage. Bathocuproine, a Cu(I)-specific chelator, and catalase inhibited the DNA damage, whereas free hydroxyl radical scavengers did not. The order of DNA cleavage sites was thymine, cytosine > guanine residues. Examinations using an oxygen electrode and cytochrome c indicate that molecular oxygen was consumed in the reaction of alpha-tocopherol and Cu(II) and that superoxide was formed. Stoichiometry studies showed that two Cu(II) ions could be reduced by each alpha-tocopherol molecule. Electron spin resonance spin-trapping investigations were then used to demonstrate that hydrogen peroxide interacts with Cu(I) to generate the reactive species responsible for DNA damage, which is either the hydroxyl radical or a species of similar reactivity. These findings may be of relevance to the tumorigenic properties of the vitamin reported in the literature. However, further studies are required to establish the significance of these reactions under in vivo conditions.     

Electron transfer between bases in double helical DNA

Fluorescent analogs of adenine that selectively oxidize guanine were used to investigate photoinduced electron transfer through the DNA pi-stack as a function of reactant stacking and energetics. Small variations in these factors led to profound changes in the kinetics and distance dependences of DNA-mediated electron-transfer reactions. Values of beta, a parameter reflecting the dependence of electron transfer on distance, ranged from 0.1 to 1.0 per angstrom. Strong stacking interactions result in the fastest electron-transfer kinetics. Electrons are thus transported preferentially through an intrastrand rather than interstrand pathway. Reactant energetics also modulate the distance dependence of DNA-mediated charge transport. These studies may resolve the range of disparate results previously reported, and paradigms must now be developed to describe these properties of the DNA pi-stack, which can range from insulator- to "wire"-like.     

Effects of UV and visible radiation on DNA-final base damage

Several mechanisms are likely to be involved in the solar radiation-mediated modifications of cellular DNA. Direct excitation of DNA bases by the UVB component (290-320 nm) of solar light gives rise, mostly through oxygen independent reactions, to the formation of dimeric pyrimidine lesions including cyclobutadipyrimidines, pyrimidine (6-4) pyrimidone photoproducts and related valence Dewar isomers. In addition, photoexcitation of cytosine and guanine may lead to the formation in relatively minor yields of 6-hydroxy-5,6-dihydrocytosine and 8-oxo-7,8-dihydroguanine, respectively. A second mechanism that requires the participation of endogenous photosensitizers together with oxygen is at the origin of most of the DNA damage generated by the UVA (320-400 nm) and visible light. Singlet oxygen, which arises from a type II mechanism, is likely to be mostly involved in the formation of 8-oxo-7,8-dihydroguanine that was observed within both isolated and cellular DNA. However, it may be expected that the latter oxidized purine lesion together with DNA strand breaks and pyrimidine base oxidation products are also generated with a lower efficiency through Fenton type reactions. A more definitive assessment of these mechanisms would require further studies aimed at the identification and quantification of the different DNA photolesions including both dimeric pyrimidine photoproducts and photooxidized lesions.     

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.     

Oxidative thymine dimer repair in the DNA helix

The metallointercalator Rh(phi)2DMB3+ (phi, 9,10-phenanthrenequinone diimine; DMB, 4,4'-dimethyl-2,2'-bipyridine) catalyzed the repair of a thymine dimer incorporated site-specifically in a 16-base pair DNA duplex by means of visible light. This repair could be accomplished with rhodium noncovalently bound to the duplex and at long range (16 to 26 angstroms), with the rhodium intercalator tethered to either end of the duplex assembly. This long-range repair was mediated by the DNA helix. Repair efficiency did not decrease with increasing distance between intercalated rhodium and the thymine dimer, but it diminished with disruption of the intervening pi-stack.     

Activation of FGF receptors by mutations in the transmembrane domain

Oxidative DNA damage by a model Cr(V) complex, [CrO(ehba)2]-, with and without added H2O2, was investigated for the formation of base and sugar products derived from C1', C4', and C5' hydrogen atom abstraction mechanisms. EPR studies with 5,5-dimethylpyrroline N-oxide (DMPO) have shown that Cr(V)-ehba alone can oxidize the spin trap via a direct chromium pathway, whereas reactions of Cr(V)-ehba in the presence of H2O2 generated the hydroxyl radical. Direct (or metal-centered) Cr(V)-ehba oxidation of single-stranded (ss) and double-stranded (ds) calf thymus DNA demonstrated the formation of thiobarbituric acid-reactive species (TBARS) and glycolic acid in an O2-dependent manner, consistent with abstraction of the C4' H atom. A minor C1' H atom abstraction mechanism was also observed for direct Cr(V) oxidation of DNA, but no C5' H atom abstraction product was observed. Direct Cr(V) oxidation of ss- and ds-DNA also caused the release of all four nucleic acid bases with a preference for the pyrimidines cytosine and thymine in ds-DNA, but no base release preference was observed in ss-DNA. This base release was O2-independent and could not be accounted for by the H atom abstraction mechanisms in this study. Reaction of Cr(V)-ehba with H2O2 and DNA yielded products consistent with all three DNA oxidation pathways measured, namely, C1', C4', and C5' H atom abstractions. Cr(V)-ehba and H2O2 also mediated a nonpreferential release of DNA bases with the exception of the oxidatively sensitive purine, guanine. Direct and H2O2-induced Cr(V) DNA oxidation had opposing substrate preferences, with direct Cr(V) oxidation favoring ss-DNA while H2O2-induced Cr(V) oxidative damage favored ds-DNA. These results may help explain the carcinogenic mechanism of chromium(VI) and serve to highlight the differences and similarities in DNA oxidation between high-valent chromium and oxygen-based radicals.     

Rapid tamoxifen-induced inactivation of an estrogenic response is accompanied by a localized epigenetic modification but not by mutations

Ethidium was found to be efficiently cross-linked to DNA by glyoxal. Kinetic studies showed that the rate of the cross-linking reaction is strongly dependent on the glyoxal concentration. Comparative studies using a series of phenanthridines and acridines showed that NH2 groups at both the 2 and 7 positions on the phenanthridine ring are necessary for efficient cross-linking. Studies using synthetic polydeoxynucleotides showed that the 2-amino group of guanine is absolutely required for cross-linking. Fluorescence contact energy transfer and relative viscosity experiments showed that the cross-linked drug remains intercalated into DNA. DNA gel electrophoresis and melting studies demonstrated that cross-linked ethidium does not dissociate the DNA double helix to single strands.     

Competitive reactions of peroxynitrite with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG): relevance to the formation of 8-oxodG in DNA exposed to peroxynitrite

We have examined the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in reactions of peroxynitrite with 2'-deoxyguanosine (dG) and calf-thymus DNA. Peroxynitrite reacts with dG at neutral pH, but this reaction does not result in the buildup of 8-oxodG. We also do not find any evidence for the formation of 8-oxodG in calf-thymus DNA upon exposure to peroxynitrite. When 8-oxodG is mixed with 1000-fold excess dG and then allowed to react with peroxynitrite, about 50% of the 8-oxodG is destroyed. The preferential reaction of 8-oxodG is also evident when dG in calf-thymus DNA is partially oxidized in an Udenfriend system and then allowed to react with peroxynitrite. We suggest that 8-oxodG is not produced in peroxynitrite-mediated oxidations of dG and DNA or that it is produced but then is rapidly consumed in further reactions with peroxynitrite. Oxidized DNA bases frequently can be more oxidation sensitive than their corresponding progenitors and, therefore, may be present at] low steady-state concentrations and not represent stable markers of oxidative stress status. The importance of the 8-oxodG/peroxynitrite reaction is discussed in relation to the formation of more stable, secondary oxidation products that might be more useful markers of DNA damage.     

Oxidative damage in chemical teratogenesis

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.     

Expression of mRNA encoding IFN alpha in macrophages stimulated with Lactobacillus gasseri

The N7 of guanine is thought to be the primary target for adduct and crosslink formation between cisplatin and DNA. However, reactive sites in DNA other than the N7 of guanine may also participate in the formation of adducts with cisplatin. The possibility that these interactions arise and form DNA polymerase blocking lesions was investigated by primer extension reactions with Taq DNA polymerase. To differentiate between damage produced at relatively weak sites from those formed at the N7 of guanine, a modified DNA template was synthesised with the N7 of guanine replaced with a carbon atom. This was achieved in a PCR designed to incorporate 7-deazaguanine instead of normal guanine. The sequence specificity of cisplatin damage in the modified and unmodified DNA substrates was compared (after linear amplification) by DNA sequencing gel analysis. For concentrations of cisplatin (1 to 5 microM) that induce blocking lesions in normal DNA, no significant damage was observed in the modified DNA. This confirmed that the N7 of guanine is the major site of adduct formation in normal DNA. At higher concentrations of cisplatin (50 microM and 100 microM), lesions were found at AA dinucleotides and other novel sites in the modified DNA. These results indicate that the N7 of guanine is not required in the formation of some cisplatin adducts.     

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer

BACKGROUND: The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. RESULTS: We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. CONCLUSIONS: Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.     

Modulated growth of Saccharomyces cerevisiae by altering the driving force of the reactions of cytochrome c: Marcus' theory in vitro and in vivo

According to Marcus' theory, rates of electron transfer reactions depend parabolically on the free energy of reaction. Amino acid replacements in the electron transport protein cytochrome c produced a series of proteins which changed the free energy of reaction for cytochrome c in oxidative phosphorylation. This study shows that Marcus' theory of electron transfer can be applied to the reactions of redox-altered cytochromes c with cytochrome c1 both in vitro and in vivo. In vitro, isolation of physiologically relevant partners of cytochrome c suggests that a change in free energy of reaction of cytochrome c changes the rate of electron transfer with cytochrome bc1 complex as would be predicted by Marcus' theory of electron transfer. Furthermore, the reactivity pattern observed in vitro is paralleled in in vivo studies. In vivo the rates of growth of Saccharomyces cerevisiae, in which these alternatives have been incorporated, also are consistent with the change in free energy of the reactions of cytochrome c with cytochrome bc1 complex. This study suggests that Marcus' theory of electron transport can predict rates not only in vitro, in isolated protein-protein systems, but also in vivo, where the relative growth rates of yeast may be predicted from the in vitro results.     

Energy transfer (deazaflavin-->FADH2) and electron transfer (FADH2-->T <> T) kinetics in Anacystis nidulans photolyase

DNA photolyases catalyze the photocycloreversion of cyclobutane pyrimidine dimers. The enzyme from the cyanobacterium Anacystis nidulans contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF). The photophysical/photochemical reactions leading to DNA repair were investigated by using time-resolved and steady-state fluorescence spectroscopy. It was found that the excited singlet state of 8-HDF transfers energy to FADH2 at a rate of 1.9 x 10(10) s-1 and a quantum yield of 0.98. Using the Forster equation for long-range energy transfer and assuming random orientations of the donor and acceptor the interchromophore distance was calculated to be 15 A. The excited singlet FADH2 which forms either by energy transfer from 8-HDF or by direct absorption of a photon has a lifetime of 1.8 ns in the absence of substrate and 0.14 ns in the presence of the photodimer indicating electron transfer from the FADH2 excited singlet state to the dimer at a rate of 6.5 x 10(9) s-1 and quantum efficiency of 92%.     

In vivo induction of sister chromatid exchange in mouse bone marrow following oral exposure to commercial formulations of alpha-cyano pyrethroids

Electron transfer from the proximal heme c-559 to the primary donor P has been studied in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis in which the tyrosine residue L162 was replaced by threonine. In the wild type, when the two high-potential hemes of the tetraheme cytochrome are reduced before flash excitation, a rapid electron transfer (t1/2 = 190 ns) observed at ambient temperature disappears below 190 K. In the mutant, the reaction is partly maintained down to 8 K, leading to irreversible charge separation. The reaction rate is nearly temperature-independent between 294 K and 8 K (t1/2 approximately 450 ns). The different behavior of wild type and mutant reaction centers is attributed to differences in a network of water molecules, the freezing of which may block structural reorganizations associated with cytochrome oxidation, in the wild type but not in the mutant.