Site Saturation Mutagenesis Demonstrates a Central Role for Cysteine 298 as Proton Donor to the Catalytic Site in CaHydA [FeFe]-Hydrogenase 英文参考文献.docVIP

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Site Saturation Mutagenesis Demonstrates a Central Role for Cysteine 298 as Proton Donor to the Catalytic Site in CaHydA [FeFe]-Hydrogenase 英文参考文献.doc

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SiteSaturationMutagenesisDemonstratesaCentralRole forCysteine298asProtonDonortotheCatalyticSitein CaHydA[FeFe]-Hydrogenase SimoneMorra1,AlbertoGiraudo1,GiovannaDiNardo1,PaulW.King2,GianfrancoGilardi1, FrancescaValetti1* 1DepartmentofLifeSciencesandSystemsBiology,UniversityofTorino,Torino,Italy, 2BiosciencesCenter,NationalRenewableEnergyLaboratory,Golden,Colorado, UnitedStatesofAmerica Abstract [FeFe]-hydrogenasesreversiblycatalysemolecularhydrogenevolutionbyreductionoftwoprotons.Protonsupplytothe catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]- hydrogenases;moreoverC298isstructurallyveryclosetotheH-clusteranditisimportantforhydrogenaseactivity.Here, thefunctionofC298incatalysiswasinvestigatedindetailbymeansofsitesaturationmutagenesis,simultaneouslystudying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstratedthatefficientenzymaticturnoverwasmaintainedonlywhenC298wasreplacedbyasparticacid,despitethe structuraldiversitybetweenthetworesidues.PurifiedCaHydAC298Ddoesnotshowanysignificantstructuraldifferencein termsofsecondarystructureandironincorporation,demonstratingthatthemutationdoesnotaffecttheoverallprotein fold.C298Dretainsthehydrogenevolutionactivitywithadecreaseofkcatonlyby2-foldatpH8.0anditcausedashiftof theoptimumpHfrom8.0to7.0.Moreover,theoxygeninactivationratewasnotaffecteddemonstratingthatthemutation doesnotinfluenceO2diffusiontotheactivesiteoritsreactivitywiththeH-cluster.Ourresultsclearlydemonstratethat,in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvementinthecatalyticfunctionof[FeFe]-hydrogenasesinagreementwithanessentialroleinprotontransfertothe activesite. Citation:MorraS,GiraudoA,DiNardoG,KingPW,GilardiG,e