The Mechanism Of Electron Transfer Between Fe(?) And Cytochrome C

Microorganisms are the driving force of the biogeochemical processes on the Earth's surface.Iron cycle processes driven by microorganisms contain two important processes:Fe?III?reduction and Fe?II?oxidation,which are closely related to the cycle of carbon/nitrogen/sulfur/heavy metals element,pollutant degradation,rock weathering and soil formation.Because of the redox potential?E_h?of Fe?II?/Fe?III?is low,and the iron form is highly susceptible to environmental factors such as pH and organic matter.Although it has been studied deeply for these factors affect iron cycle chemically,its impact on microbial-driven iron cycling processes is still not clear enough.Iron minerals have been extensively studied as photocatalyst due to its unique semiconductor properties.In recent years,researchers have found that non-photosynthetic microorganisms can acquire the photogenerated electrons of semiconductor minerals to achieve their own growth.The semiconductor properties of iron minerals have gradually attracted attention in the electron transfer between microorganism and minerals.Although it was suggested that the conductivity of semiconductor minerals promotes the extracellular electron transport process of iron-reducing bacteria,it remains unclear how the semiconducting iron minerals work on the process of microbial ferrous oxidization.c-Cyts is a key protein in the electron transport chain of organisms,and it is gradually confirmed that c-Cyts involves in the extracellular electron transport in iron-oxidizing bacteria.In this thesis,the functional protein c-Cyts was used as a model protein,to study the effects of different Fe?II?forms on kinetics and thermodynamics mechanism of electron transfer between c-Cyts and Fe?II?.At the same time,the contribution of the electron transfer mechanism between c-Cyts and different forms of Fe?II?iron?free Fe?II?,organic complexed Fe?II?,absorbed Fe?II?on iron minerals?in the mixed systems were evaluated quantitatively by model analysis.This study attempts to analyze the electron transport mechanism of microbial iron oxidation from chemical and molecular perspectives.The main research contents and results are as follows:?1?The kinetics and thermodynamic mechanism of electron transfer between free Fe?II?and cytochrome c.The effects of the initial Fe?II?concentration,pH and ionic strength on the kinetics of reaction between c-Cyts and Fe?II?were investigated.It was found that the reduction rate constant of c-Cyts increases with a growing of the initial Fe?II?concentration?5-1000?M?,which ascribed to the fact that redox potential of Fe?II?/Fe?III?decline as the Fe?II?concentration increases;The growing pH?5.5-8.0?also promoted the reduction rate constant of c-Cyts.One reason is that the reducing ability of Fe²⁺increases significantly with the increase of pH indicated by electrochemical test results that the redox potential of Fe?II?/Fe?III?is greatly reduced by the slope of 0.177/pH.In addition,spectroelectrochemical results indicate that the degree of reduction of c-Cyts is extremely correlated with pH.Therefore,the effect of pH can be attributed to the involvement of H⁺in the reaction of Fe²⁺with c-Cyts.The effect of ionic strength experiments showed that the reduction rate constant of c-Cyts went down as the growing NaCl concentration.Through the correlation analysis between the kinetic rate constant and the concentration or activity of Fe?II?species,it was found that Fe²⁺activity at different ionic strengths is an important factor affecting the reaction kinetics.This study provide a new perspective on the molecular scale of enzyme-catalyzed ferrous oxidation processes and mechanisms under anaerobic conditions.?2?The mechanism and contribution of electron transfer between organic complexed Fe?II?and cytochrome c.Due to the presence of a large amount of organic matter in the environment,complexed Fe?II?is also an important energy source for iron-oxidizing bacteria.In this study,c-Cyts protein was used as a model protein to study the effect of six different organic ligands on the reaction between c-Cyts and Fe?II?.The experimental results show that the presence of organic ligands accelerates the reaction of c-Cyts and Fe?II?.The species analysis results showed that the high content of organic complexed Fe?II?,caused by the high complex constants,was positive related to the rate constants of reaction between c-Cyts and Fe?II?.The reaction rate constants of each Fe?II?specieces reacted with c-Cyts in the mixed solutions were obtained by Kintek dynamic model software.By analyzing the correlation between the complexation constant of Fe?II?with different ligands and the apparent reaction rate constant of c-Cyts and Fe?II?,it was found that the apparent reaction rate constants is closely related with the complexation constant of complexed Fe?II?and the rate constant of c-Cyts reduced by Fe?II?L species?L represents"ligand"?.Furthermore,for the ligands with similar structure,the reaction rate constants are more highly correlated with the complexation constant of the Fe?II?complex and the rate constant?k₃?of c-Cyts reduced by Fe?II?L species.The result of cyclic voltammetry?CV?experiment showed that for the Fe?II?complex with similar structural ligands,its redox potential is positively correlated with the complexation constant,indicating that the redox potential change of Fe?II?/Fe?III?caused by ligand is not the major factor to control the reaction of Fe?II?complex with c-Cyts.The reaction reorganization energy caused by different ligand structures contributed more than the Gibbs free energy??G?to the electron transfer between complexed Fe?II?with c-Cyts.this study analyzed the kinetics and thermodynamic mechanism of electron transfer between organic complexed Fe?II?and c-Cyts from the molecular structure of the ligand,and the contribution of the electronic transfer between c-Cyts and the organic complexed Fe?II?was quantitatively evaluated by kinetic model.?3?Mechanism and contribution of electron transfer between cytochrome c and Fe?II?mediated by hematite semiconductor.Due to the adsorption and unique semiconducting properties of hematite,there are more than one way of the electron transfer between c-Cyts and Fe?II?in the mixed system.In this study,we designed kinetic experiments of redox and adsorption reaction between c-Cyts and Fe?II?with different concentrations of hematite.The results show that the addition of hematite makes the reduction rate of c-Cyts increase significantly.And the higher the hematite concentration,the greater the reduction rate of c-Cyts by Fe?II?.The electron transfer rate between the adsorbed c-Cyts and the mineral/electrode was calculated by the lavrion equation.The results show that hematite promotes the electron transfer between c-Cyts and the electrode.The I-t curve of the hematite electrode in the presence of Fe²⁺or c-Cyts solution by simulating the potential of adsorbed c-Cyts or the adsorbed Fe?II?shows that the hematite has an increased ability to donate or accept electrons from the electrode.The above results indicate that hematite can mediate and promote the electron transfer between c-Cyts and Fe?II?.The redox potential changes of the adsorbed c-Cyts and the adsorbed Fe?II?on the surface of hematite were tested by cyclic voltammetry.The results show that compared with the free Fe?II?,the potential of adsorbed Fe?II?was negatively shifted;while the potential of c-Cyts change slightly.the Mott-Schottky curve test was used to obtain the flat potential in the space charge region of hematite on the its surface before and after adsorption of c-Cyts or Fe?II?.It was found that in the presence of Fe?II?and c-Cyts,the flat band potential of hematite has a negative shift and a positive shift,respectively.In the presence of Fe?II?,the electron density on the surface of hematite increases,while the surface electron density decreases slightly with c-Cyts adsorbed,which leads to the banding of hematite in the space charge layer.Through fluorescence spectroscopy and photochemical experiments,it was found that c-Cyts can be reduced by accepting the conduction electrons of hematite.Then combined with the above experimental results,it is proposed that the built-in electric fields formed by Fe?II?and c-Cyts adsorbed on the different surface site of hematite due to their different surface electron densities accelerate the electron transfer from electrons from Fe?II?to c-Cyts.This result proposed and confirmed the hematite mediated electron transfer between Fe?II?to c-Cyts thermodynamically.The effect of different semiconductor minerals on kinetics and thermodynamic mechanism of reaction between c-Cyts and Fe?II?,indicates that there is not only conduction band of hematite mediated electron transfer between c-Cyts and Fe?II?,but also the direct electron transfer between c-Cyts and adsorbed Fe?II?.Finally,the rate constants of electron transfer between c-Cyts and adsorbed Fe?II?directly or mediated by conduction band of hematite in the mixed system are obtained through the establishment of the kinetic model.The results show that both the above two way of electron transfer rate constants are higher than the reaction rate of free Fe?II?and c-Cyts for several orders of magnitude.Under light-free and anaerobic conditions,the conduction band of hematite-mediated electron transfer process of c-Cyts and Fe?II?provides a new perspective for the study of the interaction between microorganisms and minerals in natural systems.