The Purification And Properties Analysis Of HtaB

Heme transport-associated B（HtaB）of Corynebacterium diphtheriae is a surface-anchored protein in the peptidoglycan layer of the cell wall.HtaB contains a 194 amino acid and a Conserved region（CR）domain that binds heme.HtaB is an essential component of heme uptake and transport by Corynebacterium diphtheriae.The protein heme-transport-associated A（HtaA）binds hemoglobin（Hb）and absorbs Heme;HtaA transports heme to HtaB,which transfers heme to the lipoprotein Heme uptake pathway T（Hmu T）,which transfers heme to an ATP-binding cassette（ABC）transporter consisting of the permease Heme uptake pathway U（Hmu U）and the atpase Heme uptake pathway V（Hmu V）.The Hmu TUV protein complex will facilitate heme entry into bacterial cells.Firstly,the recombinant plasmid p GEX-6p-1-HtaB was transformed into（E.coli）BL21by molecular biological methods,and the soluble expression was induced by Isopropyl-β-D-thiogalactoside（IPTG）.The target protein was purified by GST affinity column and analyzed by polypropylene gel electrophoresis.apo-HtaB without heme cogroup was obtained by butanone extraction.It was found that the conformation of HtaB changed after binding to heme,and the exposure of hydrophobic cavity was reduced.Secondly,the interaction between apo-HtaB protein and hemin/heme/hematin was investigated by fluorescence spectroscopy and ultraviolet difference spectroscopy.Apo-HtaB forms a 1:1 complex with hemin/heme/hematin,and the binding constants K_a（hemin）,K_a（heme）and K_a（hematin）were 9.90×10⁷ M^-1,3.12×10⁷ M^-1and 1.11×10⁷ M^-1,respectively.Finally,the oxidative reduction of HtaB protein was studied by UV absorption spectroscopy and electrochemical methods.The HtaB protein was found to have a Soret absorption peak at 407 nm and twoα/βbands of heme at 537 nm and 624 nm.The heme iron of HtaB protein can be reduced by Na₂S₂O₄,and the UV-visible absorption peak of heme is moved to 544 and 562 nm,which is theα/βband of divalent heme.After the redox reaction between Na₂S₂O₄ and the heme bound by HtaB protein,the Fe of the heme bound by the protein changed from trivalent to divalent.After it was exposed to oxygen,the heme was re-oxidized to trivalent,and it was also proved that the heme bound by HtaB was trivalent and its redox was reversible.The electrochemical study showed that the redox peak current of HtaB protein decreased after H₂O₂ and DTT were added,and the peak potential did not change significantly.After the addition of Na₂S₂O₄,the peak current of the protein decreased,the peak potential shifted positively,and the heme bound by HtaB was reduced.HtaB protein can be proton-coupled electron transfer reaction。...