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Mapping the molecular tunnels in three n ickel- iron hydrogenases Abd ullatypo v A.V., Ts yga nko v A. A.
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HydSL, a nicke l- iro n hydro ge nase fro m Thiocapsa roseope rsic ina, is a t he r mos tab le a nd oxyge n- to le ra nt e nzyme capab le o f hyd ro ge n o xida t io n, whic h makes it a good c a nd ida te for app lica t io n in hydro ge n fue l ce lls [1]. Unfort unate ly, t he c r ysta l s tr ucture o f t his e nzyme is not ava ilab le, whic h impedes its stud y a nd pos s ib le s ite- d irec ted mod ific atio n. We deve loped a ho mo lo gy mode l o f t his two- s ub unit e nzyme us ing MODELLER packa ge [2, 3 ] with t he nicke liro n hydro ge na se fro m Allo chromat ium v inosum (PDB- e ntr y 3 MYR) as a te mp la te. To dete r mine poss ib le pa t hwa ys o f inhib itors s uc h as o xyge n a nd ca rbo n mo no xide, mapp ing of t unne ls in t he mode l wa s carr ied o ut us ing MO LE2.0 o nline p ro gra m [4]. The mini ma l rad ius o f tunne ls a nd t he bott le ne ck rad ius we re se t to 1.2 е, whic h is t he va n de r Waa ls rad ius o f t he o xyge n mo lec ule. Ot her pa ra me ters were set to de fa ult va lues; two o t her hyd ro ge nase s, i.e. t he te mp la te a nd the nicke l- iro n hydro ge na se fro m De sulf ov ibrio v ulgaris M iya zak i F (PDB- e ntr y 1 H2 A), were a lso exp lored in MOLE 2.0. The t unne ls whic h d id no t lead to t he a ctive s ite we re re mo ved. The p ic t ure o f t he t unne ls in Desulf ov ibrio hydro ge nase was q uite s imi lar to t ha t descr ibed in lite rature. The hyd ro ge nase fro m A lloch romat ium v inosum had pr inc ipa lly t he s a me s tr uc t ure o f tunne ls, e xc ept t hat t he y we re lo nge r a nd narro we r, whereas HydS L hydro ge na se fro m Thiocap sa roseoper sic ina had no t unne ls in t he s ma ll s ub unit, t hus ha ving less pos s ib le pathwa ys fo r o xyge n mo lec ules.

a

b

c

Fig . 1. Co mp ar is on of tu n n el s tru ctu re in th ree h yd ro g en as es : a ­ HydA B h y d rog enase fro m Desulfo vibrio
vu lg ari s M iy azaki F; b ­ Hy dSL hy d ro gen as e fro m Al lo ch ro ma tiu m vin o su m; c ­ Hy dSL hy d ro gen ase fro m Th io ca p sa ro seo p ersi cin a BBS . P ictures were mad e as s creen sh o ts from J mo l; red -an d-ye llow s tic ks repres en t iro n -s u lph u r clu s ters of th e s mal l s u b u nit. Th e larg e s u b u nit is pos itio n ed on top , an d th e s mall on th e b otto m of th e imag es .


We sho uld note t hat no t unne ls co uld be fo und a fte r e ner gy mini miza t io n p roced ure o n YAS ARA e ner gy min im izatio n server [5 ]. It mea ns t hat t hese t unne ls s ho uld be c losed d ur ing a lar ge time in so lut io n a nd a re ope ned s toc has t ica lly wit h a ra t her lar ge period o f osc il latio n betwee n c lose a nd ope n s tate, so thes e res ults s ho uld be p ro ved b y mo lec ula r d yna mics simu latio ns o n supe rco mp uters wit h o xyge n as a p robe mo lec ule, so t ha t t he t unne ls co uld be represe nted as t he tracks o f o xyge n mo lec ule s ins ide the e nzyme to t he ac t ive s ite. It is inte res t ing t hat t he obta ined data s how a no ve l pos it io n for pos s ib le dete r mina nts o f oxyge n to ler a nce. The da ta fro m lit erature s ho wed the d eter mina nts o f o xyge n to lera nce in t he lar ge sub unit : t he s ub stit ut io n o f two a mino ac id re s id ues in o xyge n- se ns it ive hyd ro ge nase lead to o xyge n to lera nce [6], a nd a re ver se s ubs t it ut io n in o xyge n- to le ra nt hyd ro ge nases lead to o xyge n se ns it ivit y [7]. Ho we ve r, t he HydS L hydro ge na se fro m Thiocap sa ro seopers icina ha s t he sa me a mino ac ids as the o xyge n- sens it ive hydro ge nase fro m Desulf ov ibrio v ulgaris in cor respo nd ing pos it io ns. Thus, there is a not he r dete r mina nt o f o xyge n to lera nce s it ua ted in t he s ma ll s ub unit. We gra te fully t ha nk Kaba no v Ar t yo m Va le r ye vic h, Ko ma ro v Vlad is la v M ik ha ylo vic h, Kondrat ie v Ma xim Ser ge e vic h a nd Sa mc he nko Ale xa nde r Ana to lye vic h fo r t he ir use ful ad vice a nd he lp in t he work. REF ER ENCES 1. I.N. Gogo to v, N.A. Zor in, L. T. Sereb r yako va, E.N. Kondra t ie va. The prope rties o f hyd ro ge nase fro m Thio capsa ro seopers icina (1978). Bioch im. Biophys. Acta, 523: 335- 343. 2. Sali A, Blunde ll TL. Co mpa rative prot e in mod e lling b y sa t is factio n o f sp atia l re str a int s (1993 ) J Mol Biol., 234 (3) :779- 815. 3. She n M Y, Sa li A. St atistica l po te nt ia l for ass ess me nt a nd pr ed ic t io n o f p rote in str uctures (2006 ) Prot ein Sci.,15(11 ):2507- 24. 4. Berka K, Ha nАk O, Sehna l D, Ba nАs P, NavrАtilo vА V, Ja is wa l D, Io ne sc u CM, S vobodo vА Vareko vА R, Koca J, Ot yepka M. MOLEo nline 2.0 : inte ractive web- based a na lys is o f bio macro mo le c ular c ha nne ls (2012). Nucle ic Acids Res., 40 ( W1): W222- W227 5. Krie ger E, Joo K, Lee J, Lee J, Ra ma n S, Tho mpso n J, Tyka M, Bake r D, Karp lus K. I mpro ving phys ica l rea lis m, ste reoc he mistr y, a nd s ide- cha in acc ura c y in ho mo lo gy mode ling : Fo ur app roac hes that per for med we ll in CASP8 (2009) P rot eins, 77 S uppl 9 :114- 22 6. Lero ux F, De me nt in S, Bur la t B, Cour nac L, Vo lbeda A, C ha mp S, Mart in L, Guigl iare lli B, Ber tra nd P, Fo nt ec illa- Ca mps J, Ro usse t M, LИ ger C. Exper ime nta l app roac hes to k ine t ic s o f gas diffus io n in hydro ge nase (2008 ) Proc Nat l Acad Sci U S A, 105(32):11188- 93 7. Buhrke T, Le nz O, Kra uss N, Fr iedr ic h B. O xyge n to ler a nce o f t he H2 - se ns ing [N iFe] hydro ge nase fro m Ralstonia eut ropha H16 is based o n li mit ed acces s o f o xyge n to t he ac t ive s ite (2005) J Biol Chem, 280(25) :23791- 6.