The biosynthesis of lipoic acid: a saga of death, destruction, and rebirth Air date: Wednesday, May 1, 2019, 3:00:00 PM Category: WALS – Wednesday Afternoon …
I'M TENURE TRACK AT NICHD AND
ALSO COORDINATOR OF THE
SCIENTIFIC INTEREST GROUP WHICH
IS NEWLY FORMED, WHICH IS
HOSTING TODAY'S SPEAKER.
NOW MOST OF US LEARN ABOUT
METALS IN OUR ORGANIC CHEMISTRY
COURSE, METALS ARE ABSOLVED OF
ANYTHING TO DO WITH ORGANIC AND
BY EXTENSION BIOLOGY.
AND SO AT LEAST FOR ME WHEN I
FIRST LEARNED ABOUT METALS IN
BIOLOGY, MUCH LATER, IT SEEMED
LIKE TWO DISPARATE WORLDS COMING
TOGETHER.
BUT IT TURNS OUT THAT METALS
PERFORM AMAZING FUNCTIONS IN
BIOLOGY, ABOUT 30% OF PROTEINS
BY SOME MEASURE HAVE METAL AND
WE COULD HAVE NO BETTER LEADER
THAN OUR SPEAKER TODAY TO LEND
INSIGHTS INTO THE AMAZING WORLD
OF METAL BIOCHEMISTRY AND OF
COURSE A LOT MORE.
SQUIRE COMES TO US FROM PENN
STATE, WHERE HE'S A PROFESSOR IN
THE DEPARTMENTS OF CHEMISTRY AND
BIOCHEMISTRY AND ALSO
INVESTIGATOR IN THE HOWARD
HUGHES MEDICAL INSTITUTE.
SQUIRE IS A NATIVE OF TEXAS, GOT
HIS UNDERGRADUATE IN AUSTIN
COLLEGE AND HE HAS REALLY
TRAINED WITH THE WHO'S WHO.
HIS SERIOUS INITIATION IN
RESEARCH HAPPENED OVER A SUMMER
RESEARCH STINT IN CHRIS WALSH'S
LAB WHEN CHRIS WAS STILL AT MIT
AND I GUESS THAT'S WHAT BROUGHT
HIM BACK TO HIS MIT FOR HIS
PH.D.
SUBSEQUENTLY SQUIRE DID A SHORT
POSTDOCTORAL STINT IN PARIS ON
NITRIC OXIDE SYNTHASE AND
ANOTHER POSTDOCTORAL STINT AT
THE UNIVERSITY OF WISCONSIN AT
MADISON.
SQUIRE'S RESEARCH ADDRESSES A
BROAD RANGE OF PROBLEMS IN
ENZYME MECHANISMS AND HE HAS
COLLABORATED WIDELY AND HAS
CHOSEN WHATEVER IT TECHNIQUE
IT'S BEST FIT FOR THE PROBLEM AT
HAND, INCLUDING STRUCTURAL
BIOLOGY, ADVANCED SPECTROSCOPY
IT TECHNIQUES AND CHEMICAL
SYNTHESIS.
ONE OF HIS MAIN FOCUS HAS BEEN
THE RADICAL SAM FAMILY OF
ENZYMES THAT REALLY CAPITALIZE
MIND BOGGLING AREA OF CHEMICAL
REACTIONS AND MANY OF THESE ARE
INVOLVED IN THE SINT CYST OF
NATURAL PRODUCTS AND — NATURAL
PRODUCTS WITH ANTIBACK BACTERIAL
OR
ANTICANCER PROPERTIES.
SQUIRE HAS REALLY RECEIVED TOO
MANY AWARDS TO SAY HERE.
I'M JUST GOING TO MENTION A FEW.
THE AMERICAN CHEMICAL SOCIETY
SCHOLAR AWARD, THE PRESIDENTIAL
EARLY CAREER AWARD IN SCIENCE
AND ENGINEERING, AND HE WAS
ELECTED TO THE AMERICAN ACADEMY
OF ARTS AND SCIENCES, AND JUST
YESTERDAY, MAKES ME VERY HAPPY
TO SHARE WITH YOU THAT HE WAS
ELECTED MEMBER OF THE NATIONAL
ACADEMY OF SCIENCES.
HE'S ALSO A VERY STRONG
PROPONENT OF RAISING THE
AWARENESS AND INTEREST OF
SCIENCE IN UNDERREPRESENTED
COMMUNITIES AND WORKS VERY HARD
FOR THAT, SO IT'S A REAL
PLEASURE TO HAVE HIM HERE.
THANK YOU VERY MUCH FOR COMING,
SQUIRE.
>> HI.
IT'S A REAL HONOR TO COME HERE
TO THE NATIONAL INSTITUTES OF
HEALTH TO SORT OF GIVE A REPORT
OF HOW WE'RE SPENDING TAXPAYERS'
MONEY A.
IT'S BEEN A GREAT DAY TODAY.
I'VE KNOWN A LOT OF PEOPLE HERE
FOR QUITE SOME TIME AND IT'S
BEEN GREAT INTERACTING WITH THEM
AND LEARNING ABOUT ALL OF THE
WONDERFUL SCIENCE THAT TAKES
PLACE HERE, MUCH OF WHICH I WAS
ALREADY AWARE.
SO ONE OF THE THINGS MY LAB IS
INTERESTED IN IS HOW YOU
FUNCTIONALLIZE AN UNACTIVATED
COGNITIVE CENTER, HOW DO YOU PUT
A FUNCTIONAL GROUP ON A CARBON
CENTER THAT IS NOT EASILY
FUNCTIONALLIZED BECAUSE IT
DOESN'T HAVE PKA VALUES THAT ARE
LOW ENOUGH TO DO ACID BASE TYPE
CHEMISTRY.
AND SO MUCH OF WHAT WE KNOW — I
HAVE TO LEARN HOW TO WORK THIS
LASER POINTER.
MUCH OF WHAT WE KNOW COMES FROM
ENZYMES THAT ACTIVATE MOLECULAR
OXYGEN TO GENERATE A POTENT
OXIDANT, AND THERE A NUMBER OF
THESE TYPES OF STRATEGIES, SOME
OF THE MORE COMMON ONES ARE
ENZYMES THAT HAVE HIM COFACTORS
SUCH AS CYTOCHROME P450, THERE
ARE EP SIEMS THAT CONTAIN THESE
CAR BOX LATE BRIDGED DIE NUCLEAR
IRON CENTERS SUCH AS ME THAN
MONO OXYGENASE AND OTHERS.
THERE ARE ENZYMES THAT CONTAIN A
MONO NUCLEAR IRON THAT'S BOUND
BY AN ALPHA KE TOE IT IS
GLUTARATE COFACTOR SUCH AS TAU D
AND AMONG MANY OTHERS THERE ARE
ENZYMES THAT SIMPLY HAVE A MONO
NUCLEAR IRON THAT'S BRIDGED BY A
HIS 2 CAR BOX LATE MOTIF AND A
WATER MOLECULE AS IN
ISOPENICILLIN SYNTHASE.
IN THESE CASES, TYPICALLY
MOLECULAR OXYGEN IS A SUBSTRATE
AND I CAN SORT OF ILLUSTRATE HOW
THESE ENZYMES TYPICALLY FUNCTION
BROADLY BY THE REACTION
MECHANISMS OF CYTOCHROMES P450.
SO THESE ENZYMES WILL TAKE
COFACTOR IN THE PRESENCE OF
MOLECULAR OXYGEN, A COUPLE OF
ELECTRONS AND A COUPLE OF PROTON
PROTONS, THEY'LL SPLIT MOLECULAR
OXYGEN TO GENERATE WATER AND
THIS VERY HIGH ENERGY
INTERMEDIATE CALLED COMPOUND 1,
THIS IS AN IRON 4 4 OXO SPECIES
THAT HAS THIS RADICAL ON THE
CHAIN.
THIS HAS THE OXIDIZING POWER TO
SIMPLY RIP OFF A HYDROGEN ATOM,
THAT'S H DOT, FROM THE SUBSTRATE
TO CREATE WHAT'S CALLED COMPOUND
2, SHOWN HERE, IN THIS YOU BE
SUBSTRATE
RADICAL.
THEN THIS CAN SIMPLY REBOUND A
HYDROXYL GROUP ON TO THAT
SUBSTRATE RADICAL TO GENERATE
YOUR FUNCTIONALLIZED ORGANIC
MOLECULE.
SHOWN RIGHT HERE.
ALL RIGHT?
SO THIS IS ONE OF THE WAYS IN
WHICH YOU CAN FUNCTIONALLIZE AN
UNACTIVATED CARBON CENTER WITH
OXYGEN.
BUT IF YOU LOOK IN NATURE, IT
THERE ARE A NUMBER OF BIOWILL
MOLECULES THAT HAVE, INSTEAD OF
OH OXYGEN, THEY HAVE SULFUR THAT
HAS BEEN ATTACHED TO AN
UNACTIVATED CARBON.
SO HERE IS BIOTIN, FOR EXAMPLE,
THAT'S DERIVED DIRECTLY FROM
THIS SUBSTRAIGHT, WHERE IN A
SULFUR HAS TO BE INSERTD BETWEEN
THAT CARBON AND THAT CARBON.
HERE, FOR EXAMPLE, IS LIE IS
LIPOIC
ACID, TO GENERATE THE FINAL
PRODUCT.
THERE ARE ALSO THESE MOLECULES,
MACRO MOLECULES THAT CONTAIN
METHYL THIOL GROUPS.
THIS PROTEIN IS PROTEIN S12 OF
THE BACTERIAL RIBOSOME WHERE SUL
FAR WHERE FUR
IS , THERE
ARE A VARIETY OF TRNA MOLECULES
THAT HAVE SULFUR — SULFUR
METHYL ATTACHED TO C2 OF
ADENOSINE 37.
THESE ARE A HYPERMODIFIED TRNA
SO THERE'S TYPICALLY SOME OTHER
TYPE OF GROUP AT THIS PARTICULAR
POSITION.
A ALTHOUGH SULFUR RESIDES — THE
SAME STRATEGIES ARE AVAILABLE TO
OXYGEN FOR FUNCTIONALLIZING
UNACTIVATED CARBON CENTERS ARE
NOT AVAILABLE TO SULFUR.
AND SO ONE OF THE QUESTIONS THAT
WE WANTED TO ASK MANY YEARS AGO
WHEN WE STARTED ON THIS
PARTICULAR PROJECT IS, HOW IS IT
THAT YOU FUNCTIONALLIZE AN
UNACTIVATED CARBON CENTER WITH
SULFUR.
AND ALTHOUGH WE WORK ON ALL OF
THESE PROJECTS THAT I SHOWED YOU
YOU, OUR MAIN MODEL SYSTEM HAS
BEEN THE BIOSYNTHESIS OF THIS
MOLECULE RIGHT HERE, CALLED LIE
CALLED
LIPOIC ACID.
INVOLVED IN ENERGY METABOLISM AS
WELL AS IN THE BREAKDOWN OF
CERTAIN AMINO ACIDS AND THESE
INCLUDE THE PYRUVATE
DEHYDROGENASE COMPLEX, THE
BRANCHED CHAIN COMPLEX, THE
GLYCINE CLEAVAGE SYSTEM AND IN
BACTERIA THERE'S ACETOIN
DEHYDROGENASE.
IT'S ALMOST ALWAYS TED TETHERED
TO A
SPECIFIC RESIDUE LOCATED ON ONE
OF THE SUBUNITS OF THESE
COMPLEXES, AND WE TYPICALLY CALL
THESE SUBUNITS LIPOYL CARRIER
PROTEINS.
SPECIFIC RESIDUE IN A DN LINKAGE
AND THIS GENERATES THIS LONG
SORT OF SWINGING ARM THAT ALLOWS
THE CARRIER PROTEIN TO MEDIATE
THE TRANSFER OF INTERMEDIATES
FROM ONE PARTICULAR SUBUNIT IN
THE COMPLEX TO OTHER SUBUNITS OF
THE PARTICULAR COMPLEX.
ALTHOUGH WE'VE KNOWN FOR QUITE
SOME TIME HOW LIPOIC ACID
FUNCTIONS IN THESE COMPLEXES,
IT'S ONLY RELATIVELY RECENTLY
THAT WE'VE BEGUN TO UNDERSTAND
HOW THIS MOLECULE IS
BIOSYNTHESIZE ED EVEN THOUGH
IT'S
FAIRLY SIMPLE IN ITS STRUCTURE.
SOME OF OUR FIRST HINTS CAME
FROM PIONEERING STUDIES FROM THE
UNIVERSITY OF ILLINOIS, AND WHAT
JOHN TAUGHT US IS THAT IN
E. COLI, THERE ARE TWO PATHWAYS
FOR BUILDING THE LIPOYL
COFACTOR.
THERE'S A PATHWAY THAT HE CALLS
THE EXOGENOUS PATHWAY, AND THE
EXOGENOUS PATHWAY, FREELY POE IK
ACID CAN BE TAKEN UP, IT CAN BE
ACTIVATED BY A BYE FUNCTIONAL
ENZYME CALLED LPLA WHICH A
ACTIVATES THE CARBOYXLATE GROUP
VIA ATP TO GIVE THIS AMP
INTERNEEDED YACHT SHOWN RIGHT
HERE, AND THEN ITS SECOND
ACTIVITY IS TO TRANSFER FROM
LIP-A TO A CARRIER PROTEIN SHOWN
RIGHT HERE AND THAT GIVES YOU
YOUR COFACTOR.
SO THAT'S LIPOIC ACID BOUND TO
THE CARRIER PROTEIN IN ITS
LINKAGE.
JOHN ALSO TAUGHT US THAT THERE'S
A SECOND PATHWAY BY WHICH
ORGANISMS CAN SYNTHESIZE THEIR
OWN LIPOIC ACID DE NOVO.
THIS PATHWAY IS AN OFFSHOOT OF
FATTY ACID BIOSYNTHESIS WHICH
TAKES PLACE ON A SMALL PROTEIN
CALLED THE ACYL CARRIER PROTEIN.
AS YOU KNOW IN FATTY ACID,
BIOSYNTHESIS, YOU BUILD UP FATTY
ACID TWO CARBONS AT A TIME SO
WHEN YOU GET TO A SPECIES THAT
HAS EIGHT CARBONS, THAT BECOMES
A SUBSTRATE FOR THIS PROTEIN,
AND THAT PROTEIN WILL TRANSFER
THE EIGHT CARBON CHAIN TO YOUR
LOIP OIL
LIPOYL CARRIER PROTEIN TO GIVE
YOU THIS SPECIES RIGHT HERE.
AND IN THE VERY LAST STEP OF THE
PATHWAY, THIS PROTEIN CALLED
LIPOYL SYNTHASE, WE ABBREVIATE
LIP-A, WILL CATALYZE THE
ATTACHMENT OF SULFUR HERE AND
SULFUR THERE TO GIVE YOU YOUR
INTACT LIPOYL COFACTOR ON YOUR
LIPOYL CARRIER PROTEIN.
OKAY?
SO WHAT WE KNOW ABOUT THE
BIOSYNTHESIS OF LIPOIC ACID
BEFORE WE ACTUALLY ENTER INTO
THIS AREA, CAME PRIMARILY FROM
IN VIVO FEEDING STUDIES BY
RONALD PERRY, WHO WAS AT RICE
UNIVERSITY AFTER LEAVING
VIRGINIA TECH.
AND WHAT HE TAUGHT US IS THAT
FREE OCTANOIC ACID CAN SERVE AS
A PRECURSOR TO LIPOIC ACID IN
VIVO.
IT WAS CONVERTED WITHOUT ANY
TYPE OF DEGRADATION.
HE ALSO SHOWED THAT
TRANSFORMATION INVOLVES THE
REMOVAL OF ONLY TWO HYDROGEN, SO
OH TO MAKE LIPOIC ACID, YOU ONLY
NEED TO REMOVE HYDROGENS AT
C6 AND HYDROGENS AT C8.
AND SO THAT MEANS THAT THERE
PROBABLY NO DESATURATED
INTERMEDIATES THAT WOULD INCLUDE
DOUBLE BONDS ADJACENT TO THESE
PARTICULAR CARBONS.
SO THERE ARE NO REMOVAL OF
HYDROGENS FROM C5 AND C7.
AND I'M SURE ALL THE
ENZYMOLOGISTS WILL APPRECIATE
HERE THAT CARBON 6 IS PRO CHIRAL
AND ENZYME CAN DISTINGUISH THESE
TWO HYDROGENS IN ITS ACTIVE
SITE.
AND HIS TIER YAL CHEMICAL
STUDIES SHOW THAT IT'S THE PRO-R
HYDROGEN THAT'S REMOVED, SHOWN
RIGHT HERE, BUT THAT THE SULFUR
GOES IN WITH INVERSION OF
CONFIGURATION.
ALL RIGHT?
SO BASED ON THAT INFORMATION,
BOB WHITE, WHO WAS AT VIRGINIA
TECH, OR IT MAY HAVE BEEN RICE
AT THE TIME, SUGGESTED THAT
PERHAPS YOU MAKE LIPOIC ACID IN
THIS FASHION.
THE FIRST THING YOU DO IS USE
ONE OF THOSE P450-TYPE
MECHANISMS I SHOWED YOU ON THE
FIRST SLIDE TO MAKE OXYGENATED
INTERMEDIATES.
THEN HE SUGGESTED THAT YOU MIGHT
THEN ACTIVATE THOSE HYDROXYL
GROUPS AND THEN DISPLACE THE
ACTIVATED HYDROXYL GROUP WITH
SOME SORT OF SULFUR NUCLEOFILE.
AND THE GREAT THING ABOUT THAT
PROPOSAL IS THAT THESE P450-TYPE
REACTIONS ARE KNOWN TYPICALLY
TO SO THE GREAT THING IS THE SN2
DISPLACEM
ENT BY SULFUR
NUCLEOFILE WOULD GIVE YOU IT
THIS CONVERSION OF CONFIGURATION
THAT YOU'RE EXPECTING AT CARBON
6.
SO HE SYNTHSEISED SYNTHESIZED A
NUMBER OF
POSSIBLE INTERMEDIATES AND HE
PED ALL OF HE
FED ALL OF THESE TO E. COLI AND
FOUND NONE OF THEM WERE
CONVERTED TO LIPOIC ACID
SUGGESTING THAT THIS PATHWAY IS
NOT VIABLE.
BY CONTRAST, HE SHOWED THAT
8 WILL WILL WILL WILL —
6 MERCAPTOOCTONOIC ACID 10 TIMES
MORE POORLY.
SO THOUGH THEY WERE POTENTIAL
THEY WERE NOT GREAT
INTERMEDIATES IN THIS PATHWAY.
SO WHEN WE BEGAN TO WORK ON THIS
PROJECT, THERE WERE TWO MAIN
QUESTIONS THAT WE WANTED TO
ADDRESS.
THE FIRST QUESTION OF COURSE IS
HOW ARE WE GOING TO CLEAN THESE
HYDROGEN CARBON BONDS, FOR US TO
REMOVE PRO TO PROTONS AND THE
$64,000
QUESTION IS WHERE ARE THE
SULFURS COMING FROM.
SO THOSE ARE THE TWO MAIN
QUESTIONS THAT I WANT TO
ADDRESSED TO.
INTO SH SO IT WAS MICHAEL M
MARLETTA AND HIS CO-WORKERS THAT
FIRST CHARACTERIZED THIS ENZYME
AND HE SHOWED THAT LIPOYL
SYNTHASE BELONGED AT THAT TIME
TO AN EMERGING SUPER FAMILY OF
ENZYMES CALLED RADICAL
ADENOSYLMETHIONINE.
ANY TIME YOU NEED TO ADD A
METHYL GROUP TO SOMETHING, MOST
OF THE TIME IT'S GOING TO COME
FROM ADENOSYLMETHIONINE.
BUT IN THIS SUPER FAMILY OF
ENZYMES, ADENOSYLMETHIONINE
BINDS IN CONTACT WITH THE FOUR
IRON FOUR SULFUR CLUSTER THAT
ALL OF THESE ENZYMES ABSOLUTELY
HAVE, AND THIS WAS SHOWN BY
BRODERICK AND HOFFMAN, THIS
BINDING MOTIF, AND WHEN THAT
IRON SULFUR CLUSTER IS IN A
REDUCED STATE, IT CATALYZES THE
CLEAVAGE OF THIS FIVE PRIME
CARBON SULFUR BOND TO GIVE YOU
METHIONINE, WHICH IS BASICALLY A
SPECTATOR IN THE REACTION, BUT
ALSO THIS FIVE PRIME DEOXYADEN
SILL RADICAL.
FOR THOSE OF YOU WHO ARE
FAMILIAR AND REMEMBER ADENE.
SO — IT'S THE SAME IN DEPENDENT
ENZYMES. SO WE KNOW THIS FIVE
PRIME RADICAL IS POTENT ENOUGH
AS AN OXIDANT TO RIP OFF
HYDROGEN ATOMS EVEN FROM
UNACTIVATED CARBON CENTERS.
SO THAT ANSWERS THE QUESTION OF
MOST LIKELY HOW ARE WE REMOVING
THE HYDROGEN ATOMS, BUT AGAIN,
THE BIG QUESTION IS WHERE ARE
THE SULFUR ATOMS COMING FROM.
IT TURNS OUT AT PENN STATE, I
HAVE A LOT OF AWESOME
COLLABORATORS AND COLLEAGUES AT
PENN STATE BUT ONE OF THESE GUYS
IS A MOSS BAR SPEC IT T ARE
ROSCOPIST, IT ALLOWS YOU TO
QUALIFY THE DIFFERENT SPECIES OF
IRON IN A SAMPLE.
SO WORKING WITH CARSTEN, WE
SHOWED THAT AT THAT TIME, UNLIKE
ALMOST ALL OTHER RADICAL SAM
ENZYMES, LIPOYL SYNTHASE
CONTAINED NOT JUST 1 4 IRON
SULFUR INCLUDES TORE BUT IT
CONTAINED A SECOND ONE, THIS IS
IT THIS CLUSTER THAT IS LIGATED
BY CYSTINES IN A CX4CX5 MOTIF.
THIS MOTIF RIGHT HERE IS FOUND
ALMOST UNIVERSALLY IN RADICAL
SAM ENZYMES.
THIS MOTIF RIGHT HERE IS FOUND
ALMOST ONLY IN LIPOYL SYNTHASES.
SO WE DETERMINED THAT THESE
ENZYMES HAVE TWO 4 IRON 4 SULFUR
CLUSTERS.
THIS CLUSTER, YOU NEED TO
GENERATE YOUR RADICAL.
THIS CLUSTER, WE HAD NO IDEA
WHAT ITS FUNCTION WOULD BE IN
THIS REACTION.
AROUND 2004, CATHY DRENNAN — WE
WERE AT THE WHITE HOUSE AND
CATHY SAID I REALLY WOULD LIKE
TO TRY TO DO THE STRUCTURE OF
LIPOYL SYNTHASE.
WE WERE OF COURSE EXCITED TO
WORK WITH SOMEBODY OF HER
CALIBER.
BUT LET ME TELL YOU, IT WASN'T
EASY.
SO AFTER ABOUT 10 YEARS, TURNS
OUT THAT THE SECRET TO GETTING
THE STRUCTURE WAS GETTING THE
RIGHT HIGH SCHOOL STUDENT.
AND SO THIS GUY RIGHT HERE,
MARTIN MCLAUGHLIN, WAS A HIGH
SCHOOL STUDENT THAT CAME TO WORK
FOR ME, HE WAS JUST SUPER
EXCITED ABOUT SCIENCE, LET ME
TELL YOU THAT, AND MARTIN WAS
ABLE TO SCREEN PROTEINS, OF
COURSE, AN AROBE LICK IN THE
GLOVE BOX BECAUSE ALL OF THESE
PROTEINS HATE OXYGEN TO COME UP
WITH CONDITIONS UNDER WILLY OIL
SYNTHASE FOR MYCOBACTERIUM
TUBERCULOSIS.
ONCE HE GOT THOSE CRYSTALS, IT
WAS TIME FOR HIM TO GO OFF TO
COLLEGE, AND HE SAID, YOU KNOW,
SQUIRE, I JUST GOT ACCEPTED TO
MIT.
AND GUESS WHAT, CATHY SAID I
COULD WORK IN HER LAB.
CAN I TAKE THESE CRYSTALS WITH
ME?
AND I SAID, MARTIN, SURE, I
MEAN, IN FACT, CATHY AND I HAVE
A COLLABORATION, AND SO AS AN
UNDERGRADUATE AT MIT, HE SOLVED
THE CRYSTAL STRUCTURE OF LIPOYL
SYNTHASE.
SO QUICKLY I'LL JUST GO OVER THE
DIFFERENT DOMAINS IN THE
STRUCTURE.
THE STRUCTURE CONSISTS MAINLY OF
THREE DIFFERENT DOMAINS.
IN GREEN RIGHT HERE IS THE ALPHA
6 BETA 6 PARTIAL TEM BARREL.
THAT DOMAIN HOUSES THE IRON
SULFUR CLUSTER THAT BINDS TO S
ADENOSYLMETHIONINE, THAT'S SHOWN
DOWN HERE, AND SO THAT DOMAIN IS
RESPONSIBLE FOR GENERATING THE
RADICAL.
THIS DOMAIN RIGHT HERE IN TEAL
CONTAINS THE CYSTINES THAT BIND
TO THAT SECOND IRON SULFUR
CLUSTER, WHICH WE CALL THE
AUXILIARY CLUSTER, AND THEN THIS
LAST DOMAIN IS SORT OF THIS
ALPHA HELIX RIGHT HERE THAT KIND
OF FOLDS OVER THE AK SITE TO
SORT OVER THE ACTIVE SITE TO
SORT OF PROTECT IT.
NOW, IF YOU LOOK CLOSELY AT THE
DOMAIN THAT CONTAINS THE
CYSTINES THAT BIND TO THE IRON
SULFUR CLUSTER, THERE WAS A
PARTICULAR SURPRISE.
THE AUXILIARY CLUSTER IS BOUND
BY THREE CYSTINES AND ONE SERINE
LIGAND.
THEY'RE PREDICTED TO CONTAIN
THEIR PROTONS WHEN THEY'RE
LIGATED TO THE OXYGEN, UNLIKE
CYSTINES THAT ACT AS THIOLATES.
SO WE THINK THAT THIS CLUSTER IS
SET UP NOT TO BE STABLE.
OKAY?
BASED ON WHAT WE SAW.
NOW WE PREDICTED THAT WHILE WE
DON'T KNOW WHERE THE SULFURS ARE
COMING FROM, BUT LET'S JUST RUN
A REACTION AND SEE WHAT HAPPENS,
RIGHT?
AND SO ON THIS PARTICULAR SLIDE,
ANY TIME YOU SEE — AND LATER IN
THE TALK, ANY TIME YOU SEE A
DASHED LINE RIGHT HERE, THAT
REPRESENTS THE CONCENTRATION OF
ENZYME THAT WE HAVE IN OUR
ASSAY.
SO YOU CAN SEE.
SO IF WE RUN A LIPOYL SYNTHASE
ASSAY, THIS IS WHAT HAPPENS.
IN RED, YOU SEE RELATIVELY
RAPID, AND I SAY RELATIVELY
BECAUSE THIS AIN'T CARBONIC
ANHIGH DRAIS, LET ME TELL YOU
THAT.
RELATIVELY RAPID FORMATION OF 6
ACID RIGHT HERE AND THEN SLOW
DECAY AND THEN YOU SEE FORMATION
OF LIPOIC ACID SHOWN RIGHT
THERE.
THE INTERESTING THING IS THAT
THE AMOUNT OF PRODUCT THAT WE
GET BACK BASICALLY EQUALS THE
AMOUNT OF ENZYME THAT WE HAVE IN
OUR ASSAY.
AND THERE'S NO OTHER SULFUR
SOURCE THAT WE ADD IN THESE
PARTICULAR ASSAYS.
SO THIS SUGGESTED TO US THAT THE
ENZYME IT SELF WAS CONTRIBUTING
THE SULFURS TO THE PRODUCT THE.
WE CAN ADDRESS THIS IN THIS
EXPERIMENT.
IF WE TAKE LIPOIC ACID THAT WE
PRODUCED IN MEDIA THAT CONTAINS
NATURAL ABUNDANT SULFUR, THIS IS
JUST THE CONTROL, NATURAL
ABUNDANT SULFIDE AS THE ONLY
SOURCE OF SULFUR, THEN WE
ISOLATE THE PROTEIN, OF COURSE
THE LIPOIC ACID THAT WE GET BACK
IS GOING TO BE AT NATURAL
ABUNDANCE, WITH I IS WHICH IS
LARGELY S32
.
BY CONTRAST, IF WE DO THE SAME
EXPERIMENT BUT NOW WE
OVERPRODUCE LIP-A IN E. COLI
CULTURED IN MEDIA CONTAINING S34
LABEL SULFIDE AS OUR ONLY SOURCE
OF SULFUR, AND WHEN WE ISOLATE
THAT PROTEIN, AND THEN ASSAY FOR
LIPOIC ACID, I THINK MY POINTER
MIGHT BE DEAD — ASSAY FOR
LIPOIC ACID, ALL THE LIPOIC ACID
IS S34 LABEL AS SHOWN RIGHT
THERE.
SO THAT TELLS US THAT THE
SULFURS ARE COMING FROM THE
PROTEIN.
NOW IF WE MIX THESE TWO SPECIES
TOGETHER IN APPROXIMATELY EQUAL
AMOUNTS, WE CAN ASK THE
QUESTION, DO BOTH SULFURS COME
FROM THE SAME POLYPEPTIDE?
OR DO YOU INSERT ONE SULFUR
USING ONE POLYPEPTIDE, ONE
ENZYME, AND THEN YOU GO AND BIND
ANOTHER POLYPEPTIDE TO INSERT
THE OTHER SULFUR.
THAT'S AN EASY EXPERIMENT TO DO.
IF THEY BOTH CAME FROM THE SAME
POLYPEPTIDE, THEN YOU WOULD GET
A DISTRIBUTION OF JUST
S32 LABELED LIPOIC ACID AND S34
LABELED LIPOIC ACID.
BY CONTRAST, IF THEY CAME FROM
DIFFERENT POLYPEPTIDES, YOU
WOULD SEE A ONE TO TWO TO ONE
DISTRIBUTION OF S32 LABELED, S32
S34 AND FULLY S34 LABELED.
SO WE CAN DO THE EXPERIMENT.
HERE'S THE EXPERIMENT WITH
LIPOIC ACID ISOLATED FROM EE.
COLI CULTURED WITH NATURAL
ABUNDANT SULFUR.
HERE'S LIPOIC ACID ISOLATED FROM
E. COLI CULTURED IN S32 LABELED
SULFIDE, AND WHEN WE MIX THEM
TOGETHER IN A I PRO,
APPROXIMATELY EQUAL
WILL AMOUNTS, WHAT YOU SEE BY
AND LARGE THE PEAK AT
220 CORRESPONDING TO NATURAL
ABUNDANCE AND THE 224
CORRESPONDING TO THE S34 LABEL
SPECIES.
SO I DON'T THINK THIS IS GOING
TO WORK.
HE TELLS ME IT DOESN'T WORK ON
THE SCREEN.
SO THAT TELLS US BOTH SULFURS
COME WITH THE POLYPEPTIDE.
SO WITH THIS INFORMATION, WE CAN
START TO PIECE TOGETHER A
MECHANISM FOR HOW THIS REACTION
MIGHT TAKE PLACE.
THIS IS THE IRON SULFUR CLUSTER
THAT BINDS TO
ADENOSYLMETHIONINE.
IN ITS REDUCED STATE, IT'S GOING
TO CAUSE THE FRAGMENTATION OF
THIS PARTICULAR BOND TO GIVE US
METHIONINE AND THIS RADICAL.
THIS 5 PRIME RADICAL IS DIRECTED
TO STEREO SELECTIVELY ABSTRACT
THE 6 PRO R HYDROGEN OF THE
SUBSTRATE SHOWN RIGHT HERE, THAT
GIVES YOU ONE EQUIVALENT WHICH
WE CAN MEASURE BY GC MS, AND
THIS SUBSTRATE RADICAL SHOWN
RIGHT HERE.
NOW LET ME PAUSE FOR JUST A
SECOND.
WHEN YOU'RE DEALING WITH IRON
SULFUR CLUSTERS, FOR EXAMPLE,
THIS IS AN IRON SULFUR CLUSTER
IN A DIE MAGNETIC PLUS
2 OXIDATION STATE, WHAT THAT
MEANS IS THAT TWO OF THE IRONS
ARE AT THE OXIDATION STATE OF
FE3 AND 2 OF THE IRONS ARE AT
THE OX STATION OF FE2.
ALL OF THE SULFURS ARE AT THE
OXIDATION OF MINUS 2, ALL RIGHT?
SO THIS RADICAL RIGHT HERE, IF
IT ATTACKS THIS SULFIDE EYE ON
IN THE CLUSTER, THE SULFA
ALREADY HAS 80 ELECTRONS AROUND
IT SO THAT ATTACKS THAT SULFIDE
THAT SULFUR HAS TO GIVE UP ONE
OF ITS ELECTRONS TO ONE OF THE
IRONS, VIA INTRA — TRANSFER.
SO I'VE COLOR-CODED THESE, THE
RED CORE SPONSDZ CORRESPONDS TO
FERRIC IR
ON
AND GREENT CORRESPONDS TO
FERROUS.
THAT GUY TURNS GREEN, AND SO IN
THE PROCESS YOU HAVE A HIGHLY
REDUCED CLUSTER THAT'S UNSTABLE,
AND THIS IRON THAT'S BOUND TO
THIS WEAK LIGAND SIMPLY FALLS
OUT OF THE SYSTEM.
OKAY?
AND THAT GIVES YOU A SPECIES
THAT LOOKS LIKE A 3 IRON
4 SULFUR CLUSTER TO WHICH YOUR
ORGANIC SUBSTRATE IS ATTACHED.
SO THAT'S THE FIRST PHASE OF THE
REACTION BY LIPOYL SYNTHASE.
THIS IS ONE OF THE MORE SIMPLE
MECHANISTIC PROBLEMS WE WORK ON
BECAUSE THE SECOND PHASE IS A
RECAPITULATION OF THE FIRST
PHASE ALMOST EXACTLY IN THIS
CASE, YOU'LL TAKE A SECOND —
GENERATE YOUR SECOND EQUIVALENT
OF 5 PRIME OX SEE
ADENOSYLMETHIONINE RADICAL, THAT
IS TARGETED TO EXTRACT THE
C8 HYDROGEN ATOM RIGHT HERE, A
SECOND PRIME, THIS RADICAL HERE
CAN ATTACK THAT SULFUR OR THAT
SULFUR, WE DON'T KNOW WHICH ONE
IS ATTACKED, ALL RIGHT, AGAIN,
YOU NEED AN ELECTRON TRANSFER
BECAUSE THE SULFUR ALREADY HAS
80 ELECTRONS AROUND IT SO THAT
TURNS ANOTHER ONE OF THESE IRONS
FROM FERRIC OH FERROUS.
THE LIPOIC ACID CAN FALL OFF
ALONG WITH THAT IRON, THAT GIVES
YOU THE PRODUCT THAT WE'RE
LOOKING FOR, AND WE POSTULATE
THAT THERE IS A 2 IRON 2 SULFUR
CLUSTER REMAINING ON THE ENZYME.
OKAY?
NOW, WE'VE TESTED THIS MODEL, WE
CAN LABEL CARBON 6 AND CARBON
8 WITH DUTERIUM AND WE CAN SEE
DUTERIUM TRANSFER INTO ADENOSINE
SO THAT'S CONSISTENT WITH OUR
MECHANISM.
IN ADDITION, WE CAN QUANTIFY THE
AMOUNT OF 5 PRIME DEOXYADENOSINE
THAT WE GET BACK VERSUS LIPOIC
ACID AND WE SEE A RATIO OF 2 TO
1.
SO THAT'S ALL CONSISTENT WITH
THIS MECHANISM THAT I HAVE SHOWN
YOU.
BUT IN ENZYMOLOGY, YOU'RE ALWAYS
LOOKING FOR INTERMEDIATE, SOME
SORT OF STABLE STATE ALONG THE
REACTION COORDINATE NA THAT YOU
THAT YOU C
AN
SORT OF ISOLATE, FINDING
INTERMEDIATES THAT YOU CAN
CHARACTERIZE AND THEN SHOW
EXPERIMENTALLY THAT THEY ARE
TRULY SPECIES THAT ARE INVOLVED
IN THE REACTION.
AND SO THE KEY INTERMEDIATE IN
THIS REACTION, OF COURSE, IS
THIS CROSS SPECIES SHOWN RIGHT
HERE.
I JUST WANT TO REMIND YOU THAT
OUR SUBSTRATE, THIS OCTAN —
THIS IS THE H PROTEIN OF THE
GLYCINE CLEAVAGE SYSTEM.
IT IS ONE OF THE LIPOYL CARRIER
PROTEINS, OF COURSE IT'S NOT
KRAWN
DRAWN TO SIZE, AND THE IRON
SULFUR INCLUDES IT TER HERE IS
CONNECTED TO ANOTHER PROTEIN
THAT I'M NOT SHOWING AT ALL.
SO WE HAVE TWO PROTEINS THAT ARE
COMING TOGETHER IN THIS
PARTICULAR SPECIES.
AND SO THE QUESTION IS, CAN WE
PROVIDE EVIDENCE FOR THIS CROSS
LINKED SPECIES, FOR ONE PROTEIN
TO BE TIGHTLY BOUND TO ANOTHER
PROTEIN VIA THIS INORGANIC CROSS
LINK.
AND OUR STRATEGY FOR DOING THIS
COMES FROM THE FACT THAT WHEN WE
THROW IN ONE EQUIVALENT OF
ADENOSYLMETHIONINE, WHEN WE DO
THE REACTION, CARBON 6 GETS THE
SULFUR SIGNIFICANTLY FASTER THAN
MAKING LIPOIC ACID.
SO THE FIRST SULFUR GOES IN MUCH
FASTER THAN THE SECOND.
SO THAT SUGGESTED TO US THAT IF
WE ADD JUST ONE EQUIVALENT OF
ADENOSYLMETHIONINE, WE CAN DRIVE
EVERYTHING INTO MAKING THIS
BECAUSE YOU DON'T HAVE THE
SECOND EQUIVALENT OF
ADENOSYLMETHIONINE TO PUT SULFUR
IN AT C8 FOR THE CLUSTER TO FALL
APART.
SO THAT'S THE EXPERIMENT THAT
WE'RE GOING TO DO.
IT TURNS OUT THAT H PROTEIN AND
LIP-A BIND TIGHTLY TOGETHER, AND
SO THEY COME OUT TOGETHER ON A
GEL FILTRATION COLUMN, BUT WE
CAN SEPARATE THESE TWO PROTEINS,
AND THIS IS JUST THE MODEL, WE
DON'T HAVE A COCRYSTAL STRUCTURE
OF THAT'S TWO PROTEINS.
WE CAN SEPARATE THESE PROTEINS
BY ANION EXCHANGE CHROME TO
CHROMATOGRAPHY.
SO HERE'S THE EXPERIMENT.
HERE I'M SHOWING YOU WHERE I
MIX — WHERE MY STUDENTS MIX
LIP-A PROTEIN BUT NO SAM, SO YOU
CAN'T DO CHEMISTRY WITHOUT SAM,
AND THEY TAKE THE MIXTURE AND
THEY SUBJECT TO ANION EXCHANGE
CHROMATOGRAPHY.
THIS IS WHERE LIP-A ELUDES AND
THIS RIGHT HERE IS WHERE THE H
PROTEIN ELUDES, OKAY?
NOW, IF WE DO THE SAME
EXPERIMENT BUT NOW WE HAVE
ADENOSYLMETHIONINE IN THE
MIXTURE, WHICH ALLOWS US TO DO
THE CHEMISTRY, HERE'S WHERE
UNREACTED PROTEIN ELUDES.
HERE'S WHERE UNREACTED SUBSTRATE
OR A LITTLE BIT OF LIPOYL
CONTAINING PRODUCT ELUDES, BUT
YOU SEE THIS BAN HERE THAT HAS
INTERMEDIATE MIGRATORY
PROPERTIES, AND THIS BAND HAS
BOTH LIPOYL SYNTHASE AND THE H
PROTEIN ASSOCIATED WITH IT.
SO WE FORMED A COMPLEX BETWEEN
THESE TWO PROTEINS, ALL RIGHT?
SO NOW WE CAN DO THIS REACTION
ON A LARGER SCALE AND WE CAN
ISOLATE THIS CROSS LINK SPECIES
IN HIGH ENOUGH YIELD AND THEN
GIVE TO CARSTEN AND SAY,
CARSTEN, YOU KNOW, WHAT DOES THE
MOSSBAUER TELL US ABOUT THE
CROSS LINK SPECIES.
SO I WON'T GO THROUGH THE
DETAILS OF THE MOSSBAUER
SPECTRA, YOU CAN ASK ME ABOUT
THAT AFTERWARDS IF YOU'RE
INTERESTED, BUT CARSTEN AND HIS
STUDENTS CAN ANALYZE THIS IN
DETAIL AND WHAT HE IT TELLS ME
IS THAT WHEN I ANALYZE YOUR
SAMPLES, I SEE BASICALLY TWO
MAJOR SPECIES AND A SMALL AMOUNT
OF ANOTHER SPECIES THAT I THINK
IS A DEGRADATION PRODUCT OF ONE
OF THESE OTHER SPECIES, BUT
IMPORTANTLY, THE TWO MAJOR
SPECIES ARE A 3 IRON 4 SULFUR
CLUSTER, WHICH WE HAD PREDICTED,
AND A 4 IRON 4 SULFUR CLUSTER TO
WHICH SOMETHING IS BOUND.
SO HE CAN TELL THAT ONE OF THESE
IRON IONS IN THE 4 IRON 4 SULFUR
CLUSTER IS BOUND BY SOMETHING
BASED ON THE MOSSBAUER
PARAMETERS.
AND WE PREDICTED THAT THAT
SOMETHING WOULD BE A SAM
MOLECULE, BECAUSE THAT WOULD BE
THE CLUSTER THAT SAM ACTUALLY
BINDS TO.
OKAY.
AND SO THE NEXT QUESTION IS,
WELL, IS THE 3 IRON 4 SULFUR
CLUSTER AN INTERMEDIATE IN THE
REACTION?
SO ALL THE ENZYMOLOGISTS IN THE
AUDIENCE CAN TELL YOU THAT YOU
CAN'T RUN AROUND YELLING THAT
YOU HAVE AN INTERMEDIATE UNLESS
YOU SATISFY TWO CRITERIA.
BILL JINX TAUGHT US THIS.
YOU HAVE TO BE ABLE TO ISOLATE
THE SPECIES, THROW IT BACK INTO
A REACTION, AND SHOW THAT IT
GOES TO PRODUCT.
THAT'S CALLED CHEMICAL
COMPETENCE.
NOT ONLY THAT, YOU HAVE TO SHOW
THAT IT GOES TO A PRODUCT WITH A
RATE THAT IS AS GOOD AS THE
OVERALL RATE OF TURNOVER.
AND THAT'S CALLED KINETIC
COMPETENCE.
SO THAT'S WHAT WE'RE GOING TO
DO.
SO WE'RE GOING TO THROW IN ONE
EQUIVALENT OF
ADENOSYLMETHIONINE, AND WE'RE
GOING TO ISOLATE THIS SPECIES
SHOWN RIGHT HERE.
NOW WE NEED TO SEPARATE THESE
TWO PROTEINS FAIRLY RAPIDLY
BECAUSE DURING THE PROCESS OF
ANION EXCHANGE CHROMATOGRAPHY,
THAT SMALL AMOUNT OF THAT
CLUSTER THAT I TOLD YOU ABOUT,
WE GET A LITTLE BIT OF
DEGRADATION BECAUSE THE PROCESS
IS SLOW, THERE'S OXYGEN THAT CAN
LEECH THROUGH, SO WE GIVE A
LITTLE BIT OF DEGRADATION.
SO IN THIS EXPERIMENT, INSTEAD
OF USING AN ENTIRE PROTEIN, WE
USE A PEPTIDE THAT CONTAINS AN
OCC TANL LICE NIL RESIDUE THAT
GETS MODIFIED.
THE PEPTIDE DOES NOT BIND
TIGHTLY TO THE PROTEIN, IT BINDS
TIGHTLY TO THE PROTEIN ONLY IF
IT'S CROSS LINKED THROUGH
CHEMISTRY.
SO WE'RE GOING TO ADD ONE
EQUIVALENT OF ADENOSYLMETHIONINE
USING THIS PEPTIDE, WE'RE GOING
TO ISOLATE THE ENZYME BY GEL
FILTRATION CROW MAAING TO FEE,
THND WE'RE GOING TO QUANTIFY
BOTH OUR LIPOYL PRODUCT AS A
FUNCTION OF TIME, ALL RIGHT?
AND SO DECREASING WHAT YOU SEE
IS THE MONOTHIOLATED SPECIES, SO
WE QUANTIFY THAT AT EACH OF
THESE TIMES AND THEN INCREASING
WITH TIME IS THE LIPOYL SPECIES.
SO THEY HAVE CON ASSISTANTS ON
THE ORDER OF .1 TO .2 PER MINUTE
AND THEAPS VERY CLOSE TO THAT'S
VERY CLOS
E TO THE
OVERALL RATE CONSTANT FOR THE
REACTION, IT'S ALMOST THE SAME.
NOW, AT THE SAME TIME, WE CAN
LOOK AT CHANGES IN THE IRON
SULFUR CLUSTER AS A FUNCTION OF
TIME, AND WE CAN DO THAT BY
MOSSBAUER.
SO WE ISOLATE THAT INTERMEET
YACHT, INTERNEED
YAD, SO THIS IS JUST THE AS
ISOLATED PROTEIN BEFORE WE DO
ANY CHEMISTRY AT ALL.
YOU SEE A NICE — INDICATIVE OF
A PLAIN 4 IRON 4 SULFUR CLUSTER.
NOW WE'RE GOING TO ADD ONE
EQUIVALENT OF SAM, THEN WE'RE
GOING TO DO GEL FILTRATION,
WE'RE GOING TO ISOLATE THE
PROTEIN AND WE'RE GOING TO
CHARACTERIZE THAT BY MOSSBAUER.
AND THAT'S WHAT THESE SPECTRA
ARE.
BY MOSSBAUER, WE SEE THAT
THERE'S A 1 TO 1 RATIO OF A
3 IRON 4 SULFUR CLUSTER, AND
ANOTHER 4 IRON 4 SULFUR CLUSTER
TO WHICH ADENOSYLMETHIONINE IS
BOUND.
THEN WE'RE GOING TO ADD BACK
REDUCTION TANT AND A SECOND
EQUIVALENT OF SAM AND WHAT WE
SEE IS THAT THIS CLUSTER RIGHT
HERE COMPLETELY DEGRADES DOWN TO
FERROUS IRON AND THIS CLUSTER
STAYS INTACT WITH THE EXCEPTION
THAT ADENOSYLMETHIONINE IS NO
LONGER BOUND.
AND WE CAN TELL THAT ALL BY THE
MOSSBAUER.
SO WE START OUT WITH TWO 4 IRON
4 SULFUR CLUSTER.
AFTER ONE EQUIVALENT OF SAM, WE
GET A 3 IRON 4 SULFUR CLUSTER
AND 4 IRON 4 SULFUR CLUSTER AND
AFTER ADDING A SECOND
EQUIVALENT, THIS CLUSTER
COMPLETELY DEGRADES.
IT GETS DESTROYED.
ALL RIGHT?
BUT THE CLUSTER IN THE RADICAL
SAM SITE STAYS INTACT.
AT THE SAME TIME, WE LOOK AT
CHANGES IN OUR ORGANIC
SUBSTRATE.
SO IN THE AS-ISOLATED ENZYME,
THE PEPTIDE SUBSTRATE AND THE
ENZYME DON'T CO-ELUDE BY GEL
FILTRATION CHROMATOGRAPHY.
IF WE ADD ONE EQUIVALENT OF SAM
FOLLOWED BY GEL FILTRATION, WE
GET THE 3 IRON 4 SULFUR CLUSTER
AND THE PEPTIDE CONTAINING A
6 THIOOCC TAN LITTLE GROUP
COELUDES WITH THE ENZYME BECAUSE
IT'S CONNECTED COVALENTLY
THROUGH THE IRON SULFUR CLUSTER.
THEN WHEN WE ADD OUR SECOND
EQUIVALENT OF SAM, OUR AUCTION
ILL RYE CLUSTER IS DEGRADED TO
FERROUS IRON AND NOW OUR PEPTIDE
CONTAINS A LIPOYL GROUP.
SO THIS WAS ALL GREAT, WE WERE
SUPER EXCITED ABOUT IT, ALL THE
SPECTROSCOPISTS FELT ABOUT IT,
BUT A PICTURE IS WORTH MORE THAN
A THOUSAND WORDS, RIGHT?
SO THE QUESTION IS, CAN WE GET A
STRUCTURE OF WHAT THIS
INTERMEDIATE LOOKS LIKE?
SO I CALLED UP MY HIGH SCHOOL
STUDENT, I SAID, MARTIN, IF WE
GIVE YOU THIS PROTEIN, DO YOU
THINK — IF WE GIVE YOU THIS
INTERMEDIATE, DO YOU THINK YOU
CAN CRYSTALLIZE IT AND SHOW US
WHAT IT LOOKS LIKE?
AND HE WAS ALWAYS VERY POSITIVE
AND HE SAID SURE, I'LL GIVE IT A
SHOT.
AND SO MARTIN SOLVED THAT
STRUCTURE, AND IT BEAUTIFULLY
RECAPITULATES ALL THE MOSSBAUER
SPECTROSCOPY.
AND LET ME JUST TELL YOU,
CARSTEN NEVER BELIEVED THAT HIS
MOSSBAUER SPEC TROS
SPECTROSCOPY WAS WRON
G
AND I NEVER BELIEVED IT EITHER.
BUT HERE'S THE RADICAL SAM
CLUSTER SHOWN RIGHT HERE.
THIS IS FROM MARTIN'S STRUCTURE.
CARSTEN HAD TOLD US THAT
SOMETHING IN THIS INTERMEDIATE
SPECIES WAS STILL BOUND TO ONE
OF THE IRONS.
WE THOUGHT IT WOULD BE
ADENOSYLMETHIONINE.
WHAT IT IS IS ACTUALLY METH
METHIONINE AFTER THE CLEAVAGE TO
SAM TOOK PLACE.
WHAT YOU SEE HERE IS AFTER THE
SULFUR 5 PRIME CARBON HAS NOW
UNDERGONE CLEAVAGE, THIS HAS
BEEN TURNED INTO A RADICAL,
OKAY, AND THEN THAT RADICAL HAS
ALREADY ABSTRACTED A HYDROGEN
ATOM, THIS IS WHERE THE 6 PRO R
HYDROGEN WOULD BE POINTING.
AND IT HAS ALREADY ABSTRACTED
THAT HYDROGEN ATOM.
INTERESTING I LY, THE RADICAL
THAT'S GENERATED AT THIS
PARTICULAR CARBON HAS DONE A
BACK ATTACK ON THE AUXILIARY
IRON SULFUR CLUSTER AND IS IT'S
CREATED THIS CARBON SULFUR BOND
RIGHT HERE.
SO REMEMBER THIS GUY IS
CONNECTED TO THE H PROTEIN AND
THE CLUSTER HERE IS CONNECTED TO
LIPOYL SYNTHASE, SO THAT'S WHY
THESE PROTEINS GET CROSS LINKED
AT THIS PARTICULAR STEP.
INTERESTINGLY, THIS PICTURE
BEAUTIFULLY EXPLAINS THE STEREO
CHEMISTRY, SO YOU ABSTRACT FROM
ONE FACE WITH YOU THE BUT THE
RADICAL DOE
S A
BACK ATTACK ON THE SULFUR SOURCE
WHICH IS THE IRON SULFUR
CLUSTER, SO THAT'S HOW YOU GET
INVERSION OF CONFIGURATION AT
CARBON 6.
NOW THE INTERESTING THING IS
THAT MARTIN IS LIKE, YOU KNOW,
DR. BOOKER, I GOTTA TRY HARDER.
ONE OF OUR IRONS IS MISSING IN
OUR CLUSTER.
I'M GOING TO KEEP GOING, MAYBE I
HAD A LITTLE OXYGEN, I'M GOING
TO TRY TO KEEP GOING SO WE CAN
HAVE A FULL 4 IRON SULFUR
CLUSTER.
WE SAID NO, MARTIN, DON'T DO
ANYTHING ELSE, MAN.
THE MOSSBAUER TOLD US IT'S JUST
SUPPOSED TO BE THREE IRONS.
SO DON'T KEEP GOING, PLEASE
DON'T KEEP GOING.
AND NATURE MADE IT LIKE THAT.
SO THERE'S A REASON WHY NATURE
GAVE THAT LAST IRON A WEAK
LIGAND.
BECAUSE IT NEEDED THAT IRON TO
LEAVE THE ACTIVE SIE. THE REASON
WHY IS IN THE NEXT STEP, ALL OF
THIS CRAP HERE IN THE MIDDLE HAS
TO LEAVE THE AK ITTIVE SITE.
THAT LEAVES, THAT LEAVES AND
THEN ANOTHER MOLECULE OF
ADENOSYLMETHIONINE BINDS.
IN THIS CASE, ONCE YOU GENERATE
THE RADICAL, THE RADICAL HERE
HAS TO ABSTRACT THE HYDROGEN
ATOM CARBON 8.
AND THEN THAT CARBON, OH!
THAT CARBON HAS TO EITHER ATTACK
THAT SULFUR OR THAT SULFUR.
WE STILL DON'T KNOW WHICH ONE
HAPPENS.
BUT IT CAN'T ACCESS THOSE
SULFURS IF THE IRON IS THERE.
AND SO NATURE ACTUALLY MADE A
CHANNEL FOR THE IRON TO FALL OUT
OF THE ACTIVE SITE SO YOU CAN DO
THIS SECOND STEP IN THE
REACTION.
ALL RIGHT.
SO ALL OF THIS PART WAS THE
EFFORTS OF SOME OF MY EARLY
PEOPLE IN THE LAB AND THEN A
GREAT GRADUATE STUDENT, NICHOLAS
L OH ONZ, HE'S ALL PART OF DEATH
AND DESTRUCTION.
HE'S WORKING FOR THE FDA NOW SO
I THINK THAT'S KIND OF BAD THAT
DEATH AND DESTRUCTION IS WORKING
FOR THE FDA, BUT THAT'S
GOVERNMENT, I GUESS.
THE NEXT PART IS REBIRTH.
AND THIS IS THE PART THAT'S DONE
BY AARON MCCARTHY IN MY LAB, A
NEWER GRADUATE STUDENT.
SO WE ASKED OURSELVES, AND MANY
OTHERS IN THE FIELD, TOTALLY
DIDN'T LIKE THIS IDEA AT ALL.
THEY DIDN'T LIKE THE IDEA THAT
YOU WOULD DEGRADE AN IRON SULFUR
CLUSTER AND HAVE A PROTEIN THAT
WOULD DO JUST ONE TURNOVER.
IN FACT WE GO TO THE ENZYMES
CONFERENCE AND THEY MAKE FUN OF
US.
YOU'RE NOT REALLY STUDYING
ENZYMOLOGY, YOU DON'T HAVE BUT
ONE TURNOVER AT THE MOST.
AND SO WE FIGURED THAT THERE
MUST BE SOME SORT OF SYSTEM THAT
CAN PUT THIS CLUSTER BACK IN
EVERY TIME YOU DO A TURNOVER.
SO WE READ A LOT OF TRACY'S
PAPERS, SHE'S ONE OF THE KEY
PLAYERS IF NOT THE KEY PLAYER IN
IRON
SULFUR CLUSTER — TOLD ME ABOUT
SOME WORK FROM BRIAN ROBINSON'S
LAB THAT THIS PROTEIN IN HUMANS,
NFU1, WAS REALLY IMPORTANT IN
ASSEMBLING CLUSTERS IN 2OXOACID
DEHYDROGENASE ENZYMES, ENZYMES
THAT HAVE LIPOIC ACID.
AND THEY SHOWED THAT MUTATIONS
IN IRON CLUSTER SCAFFOLD —
CAUSE A DEFICIENCY OF — THIS
WAS ALSO CAPITULATED IN ANOTHER
STUDY SHOWN DOWN HERE, WHERE
THEY FOUND A FATAL MITOCHONDRIAL
DISEASE ASSOCIATED WITH
DEFECTIVE NFU1 FUNCTION IN THE
MATURATION OF A SUBSET OF
MITOCHONDRIAL IRON SULFUR
PROTEINS.
SO WE ASKED OURSELVES THE
COUNTERPART OF NFU1 IN E. COLI
IS THIS PROTEIN CALLED NFUA.
IS IT POSSIBLE THAT NFUA
ACTUALLY FUNCTIONING SOMEHOW IN
RESTORATION OF THE CLUSTER IN
LIPOYL SYNTHASE.
SO HERE'S AARON MCCARTHY, SO
WHAT SHE DID WAS ISOLATE IT IN
FUA AND SHOWED AS DID OTHERS
BEFORE HER THAT IT CONTAINS A
4 IRON 4 SULFUR CLUSTER.
SHE HAS A BEAUTIFUL WILL — OF
COURSE WE HAVE CARSTEN NEXT
DOOR, WE CAN VET EVERYTHING WE
DO BY MOSSBAUER SPECTROSCOPY.
IMPORTANTLY, IF SHE MIXED NFUA
AND LIP-A TOGETHER, THEY LEWDED
AS A COMPLEX.
THIS IS SHOWN IN RED.
SHE CAN TAKE ALL OF THESE
FRACTIONS AND SHOW THAT FRACTION
A HAD BOTH FRACTIONS ASSOCIATED
WITH IT, AND THIS IS YOUR NFUA
CONTROL RIGHT HERE, SHOWN RIGHT
HERE.
SO IT'S CLEAR THAT THESE
PROTEINS MIGRATE TOGETHER.
NOW THE EXPERIMENT SHE DID THAT
JUST REALLY MADE MY, I WOULD
SAY, YEAR AND POSSIBLY MADE MY
LIFETIME, I DON'T KNOW, BUT THE
EXPERIMENT THAT SHE DID WAS TO
RUN AN ASSAY.
AGAIN HERE'S CONCENTRATION OF
PROTEIN THAT WE HAVE IN THE
ASSAY.
IN RED WE'RE LOOKING AT
FORMATION OF OUR 6 INIMMEDIATE
NDK SHOWN RIGHT THERE AND AT
BLACK YOU'RE LOOKING AT
FORMATION OF YOUR LIPOYL
PRODUCT.
YOU SEE YOUR LIPOYL PRODUCT IS
LEVELING OFF JUST BELOW THE
CONCENTRATION OF ENZYME.
AND SO AT THIS POINT, WE INJECT
IRON SULFUR CONTAINING NFUA AND
YOU SEE ACTIVITY TAKES OFF
AGAIN.
SO THAT'S TELLING US THAT NFUA
POSSIBLY CAN DONATE AN IRON
SULFUR CLUSTER TO LIP-A AFTER
TURNOVER.
SO THESE ARE SOME EXPERIMENTS
WHERE SHE'S INCLUDE ED NFUA IN
THE
REACTION FROM THE START, SO THIS
IN BLACK IS REACTION JUST WITH
LIP-A BY ITSELF, 50 MICROMOLAR
ENZYME.
HERE SHE HAS A FEW EQUIVALENTS
MORE OF NFUA, AND WE MAKE MORE
PRODUCT WHEN NFUA IS PRESENT.
THEN I ASKED, LET'S TRY TO
CONVERT — I ASKED ERIN, LET'S
TRY TO CONVERT THIS INTO SORT OF
AN ENZYMATIC CATALYTIC SYSTEM.
WHY DON'T YOU DECREASE THE
CONCENTRATION OF LIP-A AND RAISE
THE CONCENTRATION OF NFUA, AND
SO HERE'S A REACTION WHERE WE
DECREASE LIP-A CONCENTRATIONS,
THIS IS THE REACTION WITHOUT
NFUA PRESENT, AND WHEN NFUA IS
THERE, YOU SEE THIS IS ALMOST
CATALYTIC, I MEAN, IT IS
CATALYTIC, YOU'RE MAKING
MULTIPLE, MULTIPLE TURNOVERS IN
THIS PARTICULAR CASE.
AND IT TURNS OUT THAT THE RATE,
THE INITIAL RATE IF WE JUST PLOT
THE FIRST FEW DATA POINTS RIGHT
HERE, THE INITIAL RATE HAS A
LOWER LIMIT OF ABOUT .25 PER
MINUTE AND FOR RADICAL SAM
ENZYME, THAT'S PRETTY
RESPECTABLE, LET ME TELL YOU
THAT.
OKAY?
NOW THE QUESTION THAT WE WANTED
TO ASK OURSELVES IS, BASED ON
OUR HYPOTHESIS THAT WE GOT FROM
MOSSBAUER, WE SHOULD BE ABLE TO
DO ONE TURNOVER OF LIP-A AND
THEN IF NFUA IS PRESENT, WE CAN
DO MULTIPLE TURNOVERS.
SO IF WE LABEL NFUA WITH S34
LABEL SULFIDE, WHAT WE SHOULD
SEE IS WHAT EQUIVALENT OF LIPOYL
PRODUCT THAT NATURAL — FOLLOW
WITH EQUIVALENT — S34 LABEL.
SO THAT'S THE EXPERIMENT THAT
SHE DID.
SHE TAKES S34 LABEL NFUA, SHE
MIXES IT WITH LIP-A AT NATURAL
ABUNDANCE.
IN BLACK, I'M SHOWING YOU THE
PRODUCT THAT CONTAINS S32 LABEL
SULFUR.
IN RED I'M SHOWING YOU THE
PRODUCT THAT HAS S34 LABEL
SULFUR AND IN BLUE, YOU SEE A
SLOW TRICKLE IN OF PRODUCT THAT
HAS MIX S32 S34.
BUT THE IMPORTANT THING IS THAT
AGAIN, THE DASHED LINE
CORRESPONDS TO THE CONCENTRATION
OF ENZYME THAT WE HAVE PRESENT.
SO WHAT'S GOING ON IS WHEN NFUA
IS PRESENT, YOU DON'T JUST DO
ONE TURNOVER ON LIP-A WITH ITS
IRON SULFUR CLUSTER, YOU'RE ABLE
TO USE ALL FOUR SULFURS IN THAT
ONE IRON SULFUR CLUSTER.
SO WHAT'S TAKEN PLACE IS HERE'S
OUR CONCENTRATION OF ENZYME,
HERE'S OUR CONCENTRATION OF
PRODUCT, THAT TWO S32 LABEL
SULFURS
AND WE'RE MAKING TWOFOLD MORE
S — THAN OUR MODEL WOULD
PREDICT AND THEN YOU START
MAKING PRODUCT THAT HAS 2S34s
ASSOCIATED WITH IT.
THIS MIX SPECIES THAT WE SEE
COMES FROM SPURIOUS BREAK KS
DOWN OF THE CLUSTER AND
RECONSTITUTION IN THE PRESENCE
OF OUR REDUCTION DANT, DIE THIGH
NIED.
OKAY?
SO BASICALLY WHAT I WANT TO
LEAVE YOU WITH IS THAT THIS
PROTEIN MOST LIKELY IN THE
PRESENCE OF NFUA USES ALL FOUR
OF THE SULFURS IN THAT SECONDARY
IRON SULFUR CLUSTER.
SO WHAT HAPPENS IS THAT IT USES
TWO SULFURS AT THE VERY
BEGINNING, AND SOMEHOW NFUA
ALLOWS THE SECOND 2 IRON
2 SULFUR CLUSTER THAT'S LEFT
BEHIND TO MIGRATE INTO THE
CATTILY SITE WHERE IT CAN BE
USED UP, AND THEN NFUA DONATES
ITS CLUSTER TO THE PROTEIN AND
THEN YOU START RECYCLING.
SO WE'RE REALLY EXCITED ABOUT
THAT.
WE CAN RECONSTITUTE NFUA WITH
SELENIUM, IN FACT, AND WHAT YOU
SEE IS TWO EQUIVALENTS OF
S32 LABEL LIPOIC ACID FOLLOWED
BY — LIPOIC ACID.
SO WE'RE EXCITED ABOUT THAT
BECAUSE YOU CAN TRACE —
CRYSTALLOGRAPHY SO WE CAN SEE
WHERE THE SELENIUMS LIE IN THE
CLUSTER AT THESE INTERMEDIATE
STATES SO WE'RE REALLY EXCITED
ABOUT THAT.
SO LASTLY, LET ME JUST QUICKLY
LEAVE YOU WITH SOME CONCLUSIONS.
SO WHAT I'VE TOLD YOU TODAY IS
THAT LIPOYL SYNTHASE UNLIKE MOST
RADICAL SAM ENZYMES HAS TWO
4 IRON 4 SULFUR CLUSTER, ONE IS
THE RADICAL SAM CLUSTER THAT
LETS YOU GENERATE YOUR POTENT
OXIDANT TO CLEAVE THE CARBON
HYDROGEN BONDS, THE SECOND
CLUSTER WITH THE WEAK SIRINE
LIGAND IS THE SOURCE — IT GETS
DEGRADED, THE PROTEIN LOSES
ACTIVITY, THAT'S WHERE DEATH AND
DESTRUCTION COMES FROM.
WE KNOW THAT YOU PUT SULFUR IN
AT C 61ST AND WHEN YOU DO C6
1ST, WHEN YO
U DO THAT YOU
GET A CROSS LINKED SPECIES WITH
AN ORGANIC GROUP ATTACHED TO IT.
THE SUBSEQUENT STEP LEADS TO
COMPLETE CLUSTER DESTRUCTION
UNDER SINGLE TURNOVER
CONDITIONS, SO THESE ARE
CONDITIONS UNDER WHICH NFUA IS
NOT PRESENT.
WE ONLY GET ONE TURNOVER.
WE KNOW THAT THE CLUSTER CAN BE
REINSTALLED IN NUFA AND IN THE
PREBS, ALL FOUR SULFIDE IONS IN
THE AUCTION IS ILL REINCLUDES IT
TER A APPEAR TO BE AK SERRATED
TO TRANSFER TO SUBSTRATE, AND
THAT'S BEEN VERY EXCITING TO US.
WE ALSO DID SOME COMPETITION
EXPERIMENTS TO SHOW THAT CLUSTER
TRANSFER FROM NFUA TO LIP-A IS
DIRECT.
I DIDN'T SHARE THOSE WITH YOU
JUST BECAUSE OF TIME.
AND THEN LASTLY, THESE ARE THE
WONDERFUL PEOPLE WHO'VE DONE THE
WORK.
NICK AGAIN IS DEATH AND
DESTRUCTION, ERIN IS REBIRTH,
MARIA AND CARSTEN, MARIA IS A
SCIENTIST AT BRAN DICE, SHE'S A
PROFESSOR THERE.
AWESOME MOSSBAUER
SPECTROSCOPIST.
MARTIN AND CATHY DRENNAN WORKED
WITH US ON STRUCTURE, MARTIN WAS
MENTORED BY PETER GOLDMAN,
GRAT WATT STUDENT WHO WAS IN
CATHY'S LAB AT THE TIME.
THESE ARE SOME OTHER PEOPLE,
MAINLY UNDERGRADUATES AND TECH
NIRKS IN MY LAB WHO WORKED ON
THE PROJECT.
AND LASTLY, LET ME JUST — I
HAVE TO THANK FUNDING BECAUSE WE
START THIS HAD PROJECT BACK, I
KOBT KNOW, WHEN I FIRST GOT TO
PENN STATE, YOU KNOW, AND BACK
THEN, NOBODY WHO WAS STUDYING
RADICAL SAM ENZYMES HAD ANY
ACTIVITY.
JUST NO TURNOVER AT ALL.
AND I SUBMITTED MY FIRST NIH
GRANT, AND WARREN JONES WAS MY
PROGRAM OFFICER, AND IT'S HARD
FOR AN ENZYMOLOGIST TO MAKE A
LIVING WITHOUT ACTIVITY, LET ME
TELL YOU THAT.
SO THE GRANT CAME BACK AND IT
WASN'T — IT DIDN'T HAVE A
FUNDABLE SCORE, LET ME SAY THAT,
BUT IT WAS ON THE LINE.
AND WARREN TOOK A CHANCE ON US
AT THAT TIME.
AND IT'S BECAUSE OF THAT THAT WE
WERE ABLE TO GET THE MONEY THAT
WE NEEDED TO SORTD OF DO THIS
WORK AND I THINK WE MADE REALLY
IMPORTANT CONTRIBUTIONS TO OUR
UNDERSTANDING OF HOW YOU
FUNCTIONALIZE UNACTIVATED CARBON
CENTERS, AND SO NIH HAS BEEN
VERY GOOD TO ME, AND IF I DIDN'T
GET THAT GRANT, I WOULDN'T BE
HERE TODAY, LET ME TELL YOU
THAT.
SO I WANT TO THANK NIH, WE'RE
GETTING PARTIAL SUPPORT TO DO
SOME OF THE STUFF WITH — FROM
THE NATIONAL SCIENCE FOUNDATION
NOW, HHMI PAYS MY SALARY NOW AND
I'M HAPPY FOR THAT.
AND I WANT TO THANK YOU FOR YOUR
ATTENTION.
IT'S BEEN A GREAT VISIT.
[APPLAUSE]
SO THE SIRINE THAT'S SUPPOSED TO
MAKE THAT LABILE, CAN YOU — TO
A CYSTINE?
>> WE'VE DONE THOSE.
WHEN YOU MAKE A CYSTINE THERE,
ALL HELL BREAKS LOOSE, ALL
RIGHT?
SO WHAT HAPPENS IS, YOU ACTUALLY
ATTACH THE CHAIN TO THE SULFUR,
SO CARBON 6 BECOMES ATTACHED TO
THE SULFUR OF THE IRON SULFUR
CLUSTER.
BUT CARBON 8 CAN'T SWING AROUND
TO ATTACK THE OTHER SULFUR,
RIGHT?
SO IN THE PROCESS, YOU'RE STILL
GENERATING RADICALS.
WHAT HAPPENS IS THAT YOU
GENERATE WHAT LOOKS LIKE A
CYCLOPROPANE RING.
SO YOU'RE STILL DOING CHEMISTRY.
YOU GENERATE A CYCLOPROPANE RING
THAT IS RESOLVED FROM THE IRON
SULFUR CLUSTER.
AND THE PROCESS, WE GET SOME
REALLY WEIRD IRON SULFUR CLUSTER
SIGNALS IN THE EPR, S IS EQUAL
WILL TO 9 HALF CLUSTER, SO
THERE'S SOME WEIRD CHEMISTRY
GOING ON WHEN WE CHANGE A
CYSTINE.
SO IT'S CLEAR THAT YOU NEED A
LIGAND THAT CAN LEAVE IN ORDER
FOR THE REACTION TO TAKE PLACE.
>> ONE MORE QUESTION.
SO IN THE — MEDIA, YOU ALSO
HAVE THE METHIONINE BINDING TO
THE OTHER —
>> RIGHT.
>> SO YOU SAID IT ALL HAD TO
LEAVE, SO WHEN THAT LEAVES, DOES
THAT AFFECT THE OTHER ENZYME —
>> I WOULD IMAGINE NOT.
SO IN THE NEXT STEP, IT JUST
TURNS OUT WE TRAPPED IT WHILE
EVERYTHING IS THERE.
SO WE DON'T HAVE A STRUCTURE
WHERE 5 PRIME ADENOSYLMETHIONINE
HAS LEFT AND — IT MIGHT HAVE
CRYSTALLIZED.
BUT IN ORDER TO DO THE NEXT
STEP, ANOTHER EE QIF LENTD OF
SAM HAS TO COME IN AND THAT
WOULD NECESSITATE THAT WOULD
HAVE TO LEAVE.
IT'S BASED ON THAT.
>> THANKS.
>> THANKS.
A VERY NICE TALK.
>> THANK YOU.
>> I HAD A QUESTION ABOUT THE
SECOND PART OF THE TALK.
>> REBIRTH.
>> EXACTLY.
>> OKAY.
>> SO DO YOU THINK — 4 IRON
4 SULFUR CLUSTER AND WHAT'S THE
FATE OF THE REMAINING 2 IRON
2 FOR THE AUCTION AUXILIARY
CLUSTER,
RIGHT?
BECAUSE YOU ARE LEFT WITH A
2 IRON 2 SULFUR CLUSTER —
>> RIGHT.
SO IN THE PRESENCE OF NFUA, WHAT
WE THINK IS HAPPENING IS THAT
THE REMAINING 2 IRON 2 SULFUR
CLUSTER MOVES IN CLOSER TO WHERE
THE SUBSTRATE AND THE 5 PRIME DE
DEOXY — ARE.
THAT ONLY HAPPENS WHEN AN FUA IS
THERE.
SO THAT SECOND 2 IRON 2 SULFUR
CLUSTER GETS USED UP, OKAY?
SO THAT'S WHY WHEN NFUA IS
PRESENT, WE GET TWO EQUIVALENTS
OF LIPOIC ACID SO YOU HAVE FOUR
SULFURS, TWO GET USED UP FOR THE
FIRST TURNOVER, TWO GET USED UP
FOR THE SECOND TURNOVER AND THE
THIRD TURNOVER AND BEYOND, THE
NUFA.
SO WHAT WE DON'T KNOW RIGHT NOW,
WE'RE ACTIVELY INVESTIGATING, IS
WHETHER NFUA SOMEHOW CONTRIBUTES
JUST A PARTIAL — A 2 IRON
2 SULFUR CLUSTER OR WHETHER IT
WAITS FOR THE OTHER STUFF TO
MOVE OVER AND IT GIVES A FULL 4.
WE'RE TRYING TO ADDRESS THAT
RIGHT NOW BY MOSSBAUER.
SO WE'RE LOOKING AT THIS WHOSE
WHOAL PROCESS BY MOSSBAUER.
WE THINK NUFA MIGHT HAVE MORE
THAN ONE FUNCTION IN THIS
REACTION.
SO YEAH, THAT'S — YEAH.
WE DON'T KNOW.
>> HI.
DO YOU NEED ANY OTHER KINDS OF
THI OH OLLY GANDZ TO EITHER
GUESS THESE — 2 IRON 2 SULFUR
CLUSTERS TO TRANSFER?
IS THERE GLUTATHIONE IN THERE?
>> THAT'S A GOOD QUESTION.
SO WHAT I CAN TELL YOU IS WE
WILL PURPOSELY GO BACK AND
ANSWER THAT QUESTION IN OUR
STUDIES, BUT WHAT I CAN TELL YOU
IS THAT ALL OF OUR REACTIONS
HAVE AT LEAST A SMALL AMOUNT OF
DTT.
AT LEAST 100 MICROMOLAR IF NOT 1
MILIMOLAR
.
SO WE ALMOST ALWAYS HAVE IT
THIOLS PRESENT.
NOW I'M PRETTY SURE A LONG TIME
AGO, WE'VE RUN THESE REACTIONS
WITHOUT THIOLS BUT THIS WASN'T
WHEN WE HAD — WHEN WE WERE
DOING THE NUFA STUFF.
THIS WAS JUST SINGLE TURNOVER
TYPE REACTIONS.
AND WE CAN GET
SINGLE TURNOVER TO TAKE PLACE
WITHOUT THIOLS, BUT FOR THIS
2 IRON 2 SULFUR TO MOVE INTO
THE — OR WHATEVER, WE HAVEN'T
DONE THAT EXPLICITLY WHEREIN
WE'VE OMITTED THIOLS AND I THINK
IT'S A GOOD ENOUGH QUESTION THAT
I'LL TELL THEM THAT WE NEED TO
DO THAT TO MAKE SURE.
THE BOTTOM LINE IS WE REALLY
DON'T UNDERSTAND THAT PROCESS
YET AND WE'RE HOPING TO USE MASS
SPECTROMETRY TO ADDRESS IT.
BECAUSE YOU CAN LOOK AT ALL
THESE CLUSTERS BY ESI MI, MS,
AND CRYSTALLOGRAPHY.
SO WE'RE HOPING WE'LL BE ABLE TO
SHED SOME LIGHT, WE'RE
INTERESTED IN THE CHEMISTRY, SO
WE'RE INTERESTED IN EXACTLY HOW
THE CLUSTERS TRAFFICKED FROM ONE
PROTEIN, SO BOND MAKING AND
BREAKING TYPE QUESTIONS.
>> SO ONE MORE QUESTION.
IF YOU WERE TO TAKE A SULFUR
CLUSTER ON NFU AND SEPARATE IT
INTO TWO — DO YOU NEED AN
ELECTRON EXCEPTOR TO DO THAT?
>> RYE.
SO WE HAVEN'T BEEN TIEBL DO
THAT, LET ME JUST SAY FIRST OF
ALL, WE HAVEN'T BEEN ABLE TO DO
THAT EXPERIMENT.
SO IT DEPENDS ON THE OXIDATION
STATES SO IF YOU HAVE A 4 IRON 4
SULFUR CL
USTER, YOU'RE GOING
TO HAVE TWO IRON 2s AND TWO
IRON 3s.
SO IF YOU HAVE A 2 IRON 2 SULFUR
CLUSTER THEY'RE MOST STABLE IN
THEIR DIE FAIRK STATE, YOU'RE
GOING TO NEED TWO ELECTRONS IN
THAT PROCESS.
IF YOU HAD TWO MIX VALENT 2 IRON
2 SULFUR CLUSTERS OF FAIRK AND A
FERROUS, THEN YOU COULD COMBINE
THEM TOGETHER AND MAKE A FULL
4 IRON 4 SILL CLUSTER WITHOUT
ADDING SILVER CLUSTER WITHOUT
ADDING ANY ELECTRONS.
>> SO LET'S JOIN IN THANKING
SQUIRE FOR A GREAT TALK.
I WANTED TO REMIND EVERYONE
THERE'S A COFFEE RECEPTION IN
THE LIBRARY AFTERWARDS

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