1-12-10 Heme and Hemoglobin: Synthesis, Structure and Function 1. Learn pathway of heme synthesis.

Heme = protoporphyrin IX Heme is a porphyrin and the prosthetic group o! hemoglobin and myoglobin "orphyrins are heterocyclic compounds deri#ed !rom $ pyrrole rings %hey can 1& comple' (ith many metals such as Fe and )g to become metalloporphyrins and 2& be in#ol#ed in o'ygen utili*ation, transport, and storage %here are + steps to heme synthesis %he !irst and !inal three steps occur in the mitochondria, and the intermediate steps occur in the cytoplasm ,ompartamentali*ation allo(s !or control o! this mechanism %here is negati#e !eedbac- inhibition o! protoporphyrin synthesis by heme in nonerythroid tissue (liver) %he steps include: succinyl ,o. / glycine / 0delta-.1. synthetase en*yme& -2 aminole#ulinic acid -2 "34, porphobilinogen 02 .1.s&-2 H)3, hydro'ymethylbilane 0$ "34s& -2 uroporphyrinogen -2coproporphyrinogen -2 protoporphyrin / Fe -2 H5)5 0(hich negati#ely !eeds bac- in nonerythroid tissue to .1. synthetase& 2. Know subcellular localization of steps in heme synthesis. Heme is made in #irtually all mammalian tissues but is most pronounced in li#er and erythroid marro( +67 heme synthesis occurs in erythropoeitic marrow 8ucleated rbc precursors can ma-e heme #ery (ell, (hile retics ha#e much reduced ability, and adult rbcs cannot ma-e heme 167 occurs in liver In the li#er, heme is made mainly !or cytochrome "$60, catalase, mitochondrial cytochromes, and other heme en*ymes 3. know important feedback points in heme synthesis. Heme produce in non-erythroid tissues 0mainly the 1I95:& negati#ely !eeds bac- on deltaaminole#ulinate 0.1.& synthetase 4. know delta-aminolevulinic acid synthetase reaction %he !irst step o! heme synthesis in#ol#es delta-aminole#ulinic acid, located in the mitochondrion Substrates: lycine and succinyl-!o" :e;uires co!actor: vitamin #$ 0pyrido%al phosphate& <elta-.1.-synthetase condenses glycine and succinyl-,o. to !orm delta-.1. 0aminole#ulinate& %he ne't step in#ol#es the condensation o! t(o delta-.1. molecules by the en*yme "L" &ehydratase 'aka (#) *ynthase+, (hich yields porphobilino en 0"34& (hich contains the !irst pyrrole ring structure

<elta-.1. synthetase is feedback inhibited by the end product heme in nonerythroid tissue %here are t(o iso!orms to delta-.1. synthetase: "L"-*1: the =house-eeping iso!orm> that is !ound in NONerythroid tissue 4i#en its location, this is the isoform that receives feedback inhibition from heme made in the liver In nonerythroid cells, .1.-S1 is the rate limiting step in heme production "L"-*2: !ound in erythroid tissue It contains an iron response element 0I:5& not present in .1.-S1 %his iso!orm is not !eedbac- inhibited by heme, but heme does inhibit cellular acquisition of iron from transferrin %he a#ailability o! trans!errin-Fe, in turn, regulates t(o iron regulatory proteins 0I:"s& that bind to the I:5 to control .1.-S2 translation ,. know ferrochelatase reaction %he !inal terminal step o! heme synthesis in#ol#es !errochelatase, also located in the mitochondrion Substrates: protoporphyrin -. and /e Ferrochelatase combines protoporphyrin IX and Fe to !orm heme $. know eneral characteristics of two of the inherited disorders of heme biosynthesis <isorders resulting !rom de!iciencies o! speci!ic en*ymes o! the heme biosynthetic path(ay are -no(n as porphyrias %hey are mani!ested by patterns o! accumulation and e'cretion o! intermediates %he t(o main clinical mani!estations o! porphyrias are: 1 cutaneous photosensitivity: results !rom e'cess tissue porphyrins 2 defects in the nervous system: poorly understood "orphyrias are either erythropoeitic 0,5", uropophyrinogen III synthetase de!iciency& or hepatic 0.I", porphobilinogen deaminase de!iciency& con enital erythropoietic porphyria 0!0(&: porphyria o! erythropoeitic origin due to a de!iciency in uroporphyrinogen III cosynthetase, an en*yme that cycli*es linear tetrapyrrole, resulting in buildup o! uroporphyrinogen I and other porphyrins in rbc?s, bone marro(, plasma, urine and !eces ,5" clinically mani!ests as light sensiti#ity in s-in, !luorescent teeth due to accumulation o! porphoryns, and anemia %reat (ith blood trans!usion, a#oiding sunlight@use sunscreen, and I9 hematin 0a !orm o! heme& acute intermittent porphyria 0"-(&: depression o! porphobilinogen 0"34& deaminase leading to a compensatory increase in delta-.1. and porphobilinogen 1arge amounts o! both o! these compounds are !ound in the urine .I" clinically mani!ests as intermittent abdominal pains and neurological disturbances, but A07 o! a!!ected patients are clinically una!!ected 0mostly hets&B Full clinical e'pression o! .I" occurs (ith e'posure to hormones and drugs 0they induce .1. synthase in the li#er& %reat .I" (ith I9 hematin 1. know the differences between the o%y en association curves of hemo lobin and myo lobin and the si nificance of this difference. 2emo lobin: %o ma'imi*e o'ygen binding, Hb e'hibits cooperativiey #ia heme-heme interactions %here are four chains (ith each heme molecule C2 binds to the Fe2/ o! each heme

It is the !our chain structure that allo(s !or Hb to ha#e cooperati#ity and display a sigmoid binding curve )odi!ying e!!ects on the sigmoid cur#e include the #ohr effect, the !32 effect, and &(). %he 2ill !oefficient is a measure o! cooperati#ity %he higher slope o! Hb 0n21& #ersus )b 0n=1& indicates that it binds o'ygen cooperati#ely, (hile )b does not In addition to cooperati#ity, Hb changes bet(een oxy 0rela'ed& and deoxy 0taut& !orms 4yo lobin: )b is a single chain molecule that produces a rectangular binding curve !or o'ygen Signi!icance: %he e!!ect o! the sigmoid cur#e 0shi!ted to the right relati#e to the rectangular cur#e& is to enable Hb to relase more o'ygen at tissues than )b %he rectangular cur#e holds on to o'ygen more tightly 5. Know the definition of the term (,6. (,6 is the partial pressure o! o'ygen at 607 Hb or )b saturation "60 #alues are in#ersely correlated (ith a!!inity !or substrate )b has a "60=2 + mmHg, (hile Hb has a "60=2D mmHg %hus, )b has a much higher a!!inity !or o'ygen than Hb and holds on to o'ygen much tighter 7. Know the eneral functions of hemo lobin in 32 and !32 transport. Hb is a protein uni;uely adapted to bind molecular oxygen in the lungs for delivery to tissues as (ell as bringing ,C2 bac- to the lungs 16. Know the role of the heme moiety and its mechanism of bindin to hemo lobin. Heme is a prosthetic group that is composed o! !our monomer globin chains, !orming a spherical tetramer It contains !errous 0Fe2/& iron Heme !its into a hydrophobic crevice in each o! the globin chains, shielding Fe2/ !rom o'idi*ing to FeE/ 8182 and 9192 are mostly ionic and (ea- contacts 8181 and 9292 !orm many stable contacts 8192 and 8291 are !unctional (ith lateral mo#ement Fhen Fe2/ is o'idi*ed to FeE/, Hb is called methemoglobin )ethemoglobin cannot bind o'ygen Hb is not co#alently bound to globin 11. Know three allosteric modulators '2:; !32; and &()+ of hemo lobin. 2:: o'ygen a!!inity !or Hb is pH dependent, as demonstrated by the #ohr effect In the 3ohr e!!ect, H/ physically binds to Hb, shi!ting the C2 association cur#e to the right %his drop in pH !acilitates the release of o%y en at tissues

&() 02,E-diphosphoglycerate&: <"4 binds deo'y Hb bet(een t(o beta chains, thus incrasing the !" of Hb# shifting the $ohr curve to the right %he rise in "60 means that <"4 has a si nificant weakenin effect on 32 bindin affinity, allo(ing !or more o%y en release into tissues Human Hb (ithout <"4 does not properly release o'ygen to tissues at physiological conditions Fetal blood has HbF, (hich has the beta chain residue 1$E histidine changed to serine %hus, !etal blood has higher oxygen affinity to !acilitate placental transport o! o'ygen !rom maternal blood !32: both ,C2 and <"4 compete !or 9al 1, the amino terminal .. on the beta chain %hus, both ,C2 and <"4 ha#e similar al-aline 3ohr e!!ects ,C2 binds more to deo'y than o'y Hb ,C2 binds more to alpha-amino groups o! G chains than H chains ,arbamino group !orms salt bridges (ith / charged group in deo'y !orm to stabili*e the deo'y !orm <"4 can displace ,C2 binding to Hb 3asic summary: increases in any o! the three allosteric modulators 0H/, ,C2, and <"4& (ill shi!t the o'ygen dissociation to the right, raise the "60, and promote C2 dissociation !rom Hb at tissues 12. Know which lobin chains are involved in the ma<or forms of hemo lobin '2b "; 2b /; 2b "2+ and know when they are formed and their relative importance. 2b " 0adult&: normal chain composition is H2G2 )a-es up A27 total Hb .bnormal . is S 0sic-le& 2b "2: H2I2 )a-es up 2 67 total Hb .bnormal .2 is 4 2b / 0!etal&: H2G2 .bnormal !etal Hb are both H 0G$, H thalassemia& and 3arts 13. know the si nificance of 2b "1c; what it is and how it is formed. Hb.1c is the glucosylated !orm o! Hb and thus re!lects the a#erage plasma glucose concentration In normal indi#iduals, Hb.1c le#els are around E7, (hile they reach D-1J7 in diabetes mellitus patients 14. know the eneral mechanism of the o%y-deo%y interconversion in hemo lobin. In deo%y 2b, the Dth position is unoccupied 0the other !i#e iron coordination sites are !illed by $ nitrogens !rom the protoporphyrin ring and 1 nitrogen !rom the imida*ole ring o! a histidine residue& Fe is out o! the heme plane In o%y 2b, the Dth position is !illed by o'ygen C'ygen pulls the Fe into the heme poc-et, and Fe pulls the pro'imal His 0F+& ligand along (ith it %hus, the F heli' mo#es (hen o'ygen binds %here are di!!erent E< tertiary and ;uaternary structures !or o'y 0rela'ed& and deo'y 0taught& !orms %hese are t(o di!!erent allosteric states, demonstrating cooperati#ity

1,. know the si nificance and mechanism of the #ohr effect. %he #ohr effect states that at lo(er pH, hemoglobin?s a!!inity !or o'ygen decreases It in#ol#es the physical binding o! protons to Hb to promote the release o! o'ygen at tissues 8ote that in tissues, the higher ,C2 le#els are, the lo(er the pH, (hich !acilitates o'ygen release !rom Hb to o'ygen-depri#ed tissues In the lungs, as #enous blood enters, re-o'ygenation re#erses the e!!ect, releasing protons !rom Hb %his in turn releases ,C2 !rom bicarbonate dissol#ed in the blood %he 3ohr e!!ect can be obser#ed on an o'ygen binding cur#e, (hich is shi!ted right(ard (ith lo( pH %he "60 increases, indicating a drop in o'ygen a!!inity and release o! C2 to tissues 1$. know the mechanism of &() bindin to hemo lobin and its role in stabilizin the structure of a specific hemo lobin form. &() interacts (ith three positi#ely charged groups on each G chain in hemoglobin It is identical to 3"4 It stabili*es the deo'y !orm o! hemoglobin 0thereby promoting o'ygen release& <"4 is metaboli*ed by the :apoport-1uebring Shunt path(ay 11. know the mechanism by which !32 interacts with hemo lobin and stabilizes a particular conformation. ,C2 binds to Hb to !orm the carbamino group .bout 10-167 o! ,C2 is transported bound to Hb in this carbamino !orm %he rest is transported by bicarbonate ,arbamino groups !orm salt bridges (ith the deo'y !orm to stabili%e it