The Theoretical Studies On The Relevant Proton/electron Transfer Mechanisms During The Catalytic Process Of The Copper-containing Nitrite Reductase

Denitrification plays an important role in keeping the global nitrogen cycle.Nitrite?NO₂^-?is reduced to nitric oxide?NO?by the copper-containing nitrite reductases?CuNiRs?,is an important step during the process of bacterial denitrification.It is well known that the catalytic reduction of nitrite by the CuNiRs requires one electron and two protons.Over the past few years,scientists from all over the world have conducted numerous experiments and theoretical studies on the reaction mechanism of the reduction of nitrite by CuNiRs.However,the unambiguous proton transfer channels and the detailed reaction mechanism of the CuNiRs have not yet been clarified.In this paper,a series of significative researches about the catalytic mechanism of the nitrite reduced by CuNiRs,including the corresponding proton transfer channels,had been carried out by utilizing the density functional theory?DFT?methods.The main contents are showed as follows:?1?The possible catalytic mechanism of the reduction of nitrite by CuNiRs is examined in the Chapter II by using M06 function according to a series of calculation modes,which including Type-one copper?T1Cu?and Type-two copper?T2Cu?sites.Examinations confirm that the protonations of two residues,His255 and Asp98,located at the second sphere of the T2Cu site,can modulate the redox states of T1Cu and T2Cu,but cannot directly cause electron transfer from T1Cu to T2Cu.The electron hole remains at the T2Cu site when only one residue,His255 or Asp98,is protonated.However,the hole resides at the T1Cu site when both His255 and Asp98 are protonated.Then,the first protonation of nitrite takes place through indirect proton transfer from protonated His255 through the bridging H₂O and Asp98 with three protons moving together,which cannot cause the cleavage of the HO-NO bond.Subsequently,the substrate is required to obtain another proton from the reprotonated His255 through the bridging H₂O.The reprotonation of nitrite induces the generation of NO and H₂O at the T2Cu site through a special double-proton-coupled spin-exchanged electron-transfer mechanism with indirect proton transfer from His255 to the substrate,a beta-electron of T2Cu⁺shifting to the NO cation,and the remaining alpha-electron changing spin direction at the same time.?2?CuNiRs is a key enzyme in the biological conversion of nitrite to nitric oxide,which involves long-range electron/proton transfer reactions.However,details of the electron/proton transfer mechanisms during the catalytic reaction of CuNiR are still unknown.Especially,the real driving force for electron transfer from T1Cu to T2Cu is ambiguous.In Chapter III,we display the possible proton transfer steps through two putative proton channels,the first proton channel and the second proton channel,by using DFT calculations.Our examinations confirm that the driving force of electron transfer from T1Cu to T2Cu comes from a remote triple-proton synchronized transfer from Lys128 to His260 along the first proton channel or from Glu113 to an intermediate water molecule along the second proton channel,which can be termed as a remote triple-proton-coupled ET mechanism.Then,the two proton channels employ three/two steps of proton transfer to deliver the first proton to the nitrite substrate at the T2Cu site.?3?The nitrite obtains the first required proton to form the intermediate HONO in the catalytic reaction of CuNiRs.Subsequently,the HNO₂ accepts the second required proton utilizing the two proposed proton channels,and changes into NO and H₂O.But the detailed reaction mechanisms of proton transfer from the outside of the protein to the HNO₂ have not been explored.In Chapter IV,we examine the possible proton transfer steps along the two proton channels by using ONIOM calculations according to a series of protein models with HNO₂ bound to T2Cu.My examinations reveal that the first proton channel employs two steps of triple-proton synchronized transfer to deliver proton from Lys128 to His255.Then,the HNO₂ indirectly obtains a proton from His255utilizing a double-proton-coupled electron-transfer mechanism,with a beta-electron transfer from T2Cu site to the NO portion.The second proton channel delivers a proton from Glu113 to Asp98 through a triple-proton synchronized transfer mechanism and a single-proton transfer mechanism.Subsequently,proton transfer takes place from Asp98to the HNO₂ accompanied by a beta-electron transfer from T2Cu⁺to the NO cation and the remaining beta-electron of the T2Cu site changing spin direction.The analyses of the energy barriers of two proton channels reveals that the second channel is the main PT channel during the catalytic process of CuNiRs.