| Electron transfer(ET)is ubiquitous in biological systems,which is closely related to the transmission of biological signals and the realization of physiological functions.In the past decades,the behavior of ET in organisms has been deeply studied both theoretically and experimentally.However,these studies mainly focus on the long-range(Electron donoracceptor distance > 10 (?))ET dynamics,which usually occurs in the range from nanosecond to millisecond(ns-ms),while the protein solvation relaxation process usually occurs in picosecond(ps).Therefore,long-range ET is in equilibrium,and its dynamics usually shows a single exponential decay behavior,which satisfies the classical Marcus ET theory.When the distance between electron donor and acceptor becomes shorter(Electron donor-acceptor distance < 10 (?)),the ET rate will become faster,which will occur in the range from picosecond to nanosecond(ps-ns).Even when the distance is shortened to the van der Waals contact,ET dynamics will occur in femtosecond(fs).Therefore,the short-range ET will be coupled with the solvation process.Many reactions involving life activities are related to short-range ET in protein,and many long-range ET processes are essentially a combination of multiple short-range ET.Therefore,understanding the coupling process between short-range ET and environmental relaxation and the form of ET(Direct jump or tunneling)is very important for the study of short-range and long-range ET in proteins,which provides a research basis for proteins that need to use ET to achieve important functions.Flavodoxin is a kind of ET protein in bacteria.The protein contains a cofactor flavin mononucleotide(FMN)and two natural electron donors,tryptophan(W)and tyrosine(Y)within the van der Waals contact to FMN.Under light excitation,short-range ET will occur in the flavodoxin,which makes it a suitable research object of short-range ET.Ultrafast spectroscopy can be used to study the dynamic changes of atoms,molecules and biological samples in the limit time range,so as to reveal the physicochemical properties of substances.In this paper,Anabaena flavodoxin is selected as the research object,and its short-range ET is systematically studied by ultrafast spectroscopy.The main research contents are as follows:(1)The solvation dynamics of protein were studied.Firstly,W and Y near FMN in Anabaena flavodoxin were mutated to electron inert phenylalanine(F),and then the solvation process of the protein was characterized by using ultrafast fluorescence spectroscopy.Finally,by constructing the solvation correlation function and fitting the data,the solvation dynamics were obtained.The time scales are 1 ps,28 ps and 267 ps,and the data provide a quantitative basis for subsequent theoretical calculation.(2)The ET dynamics of oxidized protein were studied.Flavodoxin is oxidized in the natural state,so we first characterized the ET dynamics of the protein in the oxidized state in this chapter.Firstly,the ET behaviors in different electron donors(W and Y)of Anabaena flavodoxin were characterized by using ultrafast fluorescence spectroscopy and transient absorption spectroscopy.The forward ET(FET)occurs in 100-200 fs,and the solvent stays frozen.The back ET(BET)occurs in 1-2 ps and the vibrational cooling occurs in 3-6 ps.At the same time,the results were compared with the reported data of oxidized Desulfovibrio vulgaris(D.vulgaris)flavodoxin.We discovered that Y donor has a stronger coupling with FMN in Anabaena flavodoxin that results a faster ET rate of Y donor.Finally,the nonequilibrium ET dynamics in oxidized state were analyzed by using recently proposed nonergodic theory.The results showed the negligible outer(solvent)reorganization energy but a large inner reorganization energy.Such similar large reaction free energy and inner reorganization energy result in the minor activation energy,thus electron coupling plays an important role in ET process.(3)The ET dynamics of semiquinone protein were studied.By changing the driving force of ET reaction,the influence on ET dynamics were studied: when the protein is reduced to semiquinone state,the reduction potential becomes lower and the driving force of reaction becomes weaker,which will make the ET reaction slow down and couple with the solvation process.Firstly,the proteins were reduced to semiquinone state via photoreduction method.Then,the ET dynamics were characterized by using transient absorption spectroscopy.Both of the FET and BET processes ouccur in picoseconds,thus couple with solvation.The results were compared with those of oxidized state and those of semiquinone state of D.vulgaris flavodoxin.We discovered that the ET dynamics of Ydonor is still faster,indicating a stronger coupling between Y and FMN in Anabaena semiquinone state.Finally,the nonergodic theory was used to analyze the dynamics of nonequilibrium ET in semiquinone state.The results showed that the outer(solvent)reorganization energy is still relatively small,but can not be ignored as in the oxidized state,resulting in the stretched ET dynamics.(4)The ET dynamics of distance-dependent protein were studied.By changing the electronic coupling of ET reaction,the influence on ET dynamics were studied: when the distance between electron donor and acceptor changes,the coupling will change,which will affect ET dynamics.Firstly,the W and Y closest to FMN in Anabaena flavodoxin were mutated to F and then the designed electron donor W was introduced via the mutation method.The distance between W and FMN changed within 3-10(?) so as to change the coupling between the ET donor and the receptor.Then their ET dynamics were characterized by using transient absorption spectroscopy to explore the impact of different distances on ET behavior.The results showed that the ET dynamics of mutants varis from hundreds of femtoseconds to picoseconds and nanoseconds,basically increases with the increase of the distance from W to FMN.Because some W donors have wobbling behavior,we need to describe them with two ET decays,and we can also use the wobbling model for analysis.Finally,our study showed that the ET process is affected not only by electron coupling,solvation process and protein wobbling,but also by orbital orientation and tunneling path. |
