Single-molecule Formate Dehydrogenase Catalytic Mechanism Research

Nature has evolved a vast range of oxidoreductase which play an important role to maintain the life activities on earth.Formate dehydrogenase(FDH)is an important oxidoreductase that catalyzes the dehydrogenation of formate to form carbon dioxide through the electric coupling between FDH and NAD+.So,it is not only widely used in the regeneration of coenzyme in the enzymatic production system in industrial products but also attracts many scholars on the modification and design of FDH to initial the reverse reaction and applied in the elimination of the greenhouse effect.Although various works have investigated the conformational changes and the kinetic behavior in a catalytic process using cryo-EM,X-ray,and dynamic simulation,it is still a challenge to detected dynamic enzyme catalytic processes in real-time.However,most previous studies are based on ensemble-averaged experiments,which swamp the transient intermediate or processes involved in enzymatic catalysis.Thus,the singlemolecule technique,such as nanopore,optical tweezers,atomic force microscope,and scanning tunneling microscope break-junction(STM-BJ)offers the opportunity to monitor the reaction dynamics of the enzymes.In this paper,we construct an electrical test platform based on the STM-BJ technique to probe the interaction between FDH and NAD+and reveal the FDH catalytic reaction mechanism.The main research contents and results are as follows:First,a single molecular scale formate dehydrogenase electrical test platform is established.The enzymatic properties and kinetic parameters of formate dehydrogenase are studied,and the optimum catalytic conditions and reaction time of formate dehydrogenase is determined.The measuring method of STM-BJ applied to a singlemolecule enzyme system was optimized,in which the needle tip is mechanical erosion and covered with apiezon wax to minimize the leakage current from the solution.The result shows a molecular plateau of FDH(pink)junctions with conductance at 10^-4.28 G0,which demonstrated that we constructed the platform successfully for singlemolecule enzyme measurement.At the same time,to further verify the connection sites in the enzyme,site-directed mutation(cysteine at site 23 to serine)was carried out to construct an FDH without the two-terminal L-cysteines.However,we couldn’t collect any molecular conductance signal in the measurement suggesting that the binding through L-cysteines is essential in single-molecule measurement.Thus,the formate dehydrogenase with two cysteines can formed molecules junction of Au-molecules-Au in STM-BJ measurement.So,the single-molecule conductance measurement platform for oxidoreductase through STM-BJ has been constructed successfully,which lays a good foundation for further study.Second,the charge transport changes have been studied in the binding process of NAD+to FDH.We find that the addition of NAD+leads to the conductance enhancement to 10^-2.96 G0,which is 21 times higher than single FDH junctions(10^-4.28 G0).To verify whether the adsorption or active binding was the main reason for the significant increment,the charge transport through the assembly of FDH and another cofactor,NADP+is also examined.The phosphate group combined with the ribose made NADP+ unable to act as the cofactor of FDH in our work due to the steric hindrance and charge repulsion.Although NADP+ may also interact with the surface of FDH,the significantly lower conductance(10^-3.93 G0)increase than that of NAD+suggest that the matching of NAD+to the active center of FDH provides the better electric coupling between FDH and NAD+ with some specific amino acid residues through the H-bond.Besides,the conductance of FDH with different concentrations of NAD+(1:67227,1:571429,1:2285714,and 1:11428571)and with different pH values(3.5,6.5,8.5,10.5,and 12.5)was also measured.Combined with the results of enzyme activity,the concentration of NAD+affects both the catalytic rate and electron transfer rate of the enzyme.Third,the effect of NAD+on the electron transfer rate of FDH is investigated.To provide further insight into the electric coupling of this assembly process,DFT calculations of these structures are performed by using the B3LYP to reveal the electron density distribution of the frontier orbitals of FDH and FDH-NAD+.The calculation results show that the addition of NAD+not only affects the distribution of HOMO and LUMO but also decreased the HOMO-LUMO gap of FDH-NAD+ from 5.74 eV to 3.22 eV,suggesting NAD+ could significantly enhance charge transport.Besides,flicker noise analysis shows the transition of the transmission path from the through-space path in FDH to the through-bond path in FDH-NAD+which indicates the NAD+involved in the charge transport path through the FDH-NAD+and caused the changes of conductance.So,the three amino acid residues of R174,D282,and Y194,which interact with NAD+,are replaced by K(lysine),N(asparagine),and I(isoleucine)by using site-directed mutagenesis respectively to construct R174K-FDH,Y194I-FDH,and D282N-FDH.It is interesting to find that their enzyme activity,conductance,and binding energy were positively correlated.Thus,we can conclude that the weaken of H-bond in different sites of the NAD+binding center led to changes of binding energy and therefore make the enzymic activity various at last,which is reflected in the conductance changes due to the effect derived from binding energy.Four,the catalytic reaction process is monitored through the STM-BJ and hover model.The significant differences between FDH and FDH-NAD+in the conductance have been investigated in chapters one to three,so the conductance of FDH,FDHNAD+,FDH-NADH,and FDH-HCO2-are used as markers to probe the reaction states in the dynamic catalytic process of FDH in this chapter.The conductance of FDH,FDHNAD+,FDH-NADH,and FDH-HCO2-is 10^-2.68 G0,10^-3.04 G0,10^-4.38 G0,and 10^-4.45 G0 through the STM-Break Junction model,suggesting HCOONa do not affect the conductance of FDH.Then the catalytic reaction of FDH is further monitored,in which five transient states with recognizable conductance(named as T1,T2,T3,T4,and T5),are captured in the different experiment,suggesting the catalytic reactions isn’t affected by the excessive addition of either HCOONa or NAD+.Among them,T1,T2,and T5 are FDH-NAD,FDH-NADH and FDH probed by the makers respectively.And the two unknown states labeled as T3 and T4 may be the intermediate states captured in the reaction process(FDH-NAD+-HCO2-and FDH-NADH-CO2),Although,the intermediate states are successfully captured through the Break Junction model in the FDH catalytic reaction process,the states with conduction are discontinuous result in the lack of time parameter.So,the hover model was employed to monitor the catalytic reaction process of FDH continuously.Combined data processing results show that the hover model can not only capture the same five conductance states,but also a continuous catalytic cycle.Analysis results show that not the classical catalytic cycle(T2-T5-T1-T3-T4)in the Theorell-Chance mechanism but a new order(T2-T1-T3-T4)is the main catalytic cycle probed in the single-molecule level of the FDH reaction process.More specifically,FDH-NADH did not dissociate the NADH from its active center and combined with new NAD+at the end of the reaction,but in transition to FDH-NAD+directly.The combined result of QM/MM also explains these processes theoretically.Computational studies reveal that the bound NADH converts to NAD+directly via the hybrid transfer reaction with a lower Gibbs energy barrier.