FLAVOPROTEIN PROTOCOLS
CONTENTS UV-VISIBLE SPECTROSCOPY AS A TOOL TO STUDY FLAVOPROTEINS IDENTIFYING AND QUANTITATING FAD AND FMN IN SIMPLE AND IN IRON– SULFUR-CONTAINING FLAVOPROTEINS FLUORESCENCE ANALYSIS OF FLAVOPROTEINS POTENTIOMETRIC MEASUREMENT OF OXIDATION–REDUCTION POTENTIAL FLAVOPROTEIN KINETICS APPLICATION OF FREEZE QUENCHING TO THE STUDY OF RAPID REACTIONS IN FLAVOPROTEINS APPLICATION OF ELECTRON SPIN RESONANCE (ESR) FOR DETECTION AND CHARACTERIZATION OF FLAVOPROTEIN SEMIQUINONES CIRCULAR DICHROISM STUDIES OF FLAVOPROTEINS VIBRATIONAL SPECTROSCOPY OF FLAVOPROTEINS NMR OF FLAVOPROTEINS FLAVOPROTEIN RESOLUTION AND RECONSTITUTION FLAVOENZYME STRUCTURE AND FUNCTION: APPROACHES USING FLAVIN ANALOGUES IDENTIFICATION OF COVALENT FLAVOPROTEINS AND ANALYSIS OF THE COVALENT LINK FLAVIN SYNTHESIS AND INCORPORATION INTO SYNTHETIC PEPTIDES COMPUTATIONAL METHODS IN FLAVIN RESEARCH FLAVINS AND FLAVOENZYMES IN DIAGNOSIS AND THERAPY AND ?450 NM ARE OBSERVED (SEE FIG 2) METHOD 1 DESCRIBES A PROCEDURE TO DETERMINE THE EXTINCTION COEFFICIENT FOR A FLAVIN BOUND TO A PARTICULAR FLAVO- PROTEIN WHICH IS BASED ON THE KNOWN EXTINCTION COEFFICIENT FOR FREE FMN AND FAD (12,500 AND 11,300 M–1 CM–1, RESPECTIVELY) BINDING OF THE FLAVIN TO THE PROTEIN MOIETY ALTERS THE EXTINCTION COEFFICIENT COVERING A RANGE FROM 10,500–15,400 M–1 CM–1 (3) IN SOME CASES THE PROTEIN LOWERS THE PKA FOR THE N(3)-HYDROGEN (= 10 3 FOR FREE FLAVIN) RESULTING IN DEPROTONATION AND A LOWER EXTINCTION COEFFICIENT THIS WAS OBSERVED FOR GLYCOLATE OXIDASE (9200 M–1 CM–1, (4)) EXTINCTION COEFFICIENTS IN THAT RANGE MAY THEREFORE INDICATE THAT THE PRO- TEIN PREFERENTIALLY BINDS THE N(3)-DEPROTONATED FLAVIN FIG 1 STRUCTURE OF RIBOFLAVIN FIG 2 UV-VISIBLE SPECTRUM OF FMN IN ITS OXIDIZED AND REDUCED FORM FLAVOPROTEIN PROTOCOLS KNOWLEDGE OF THE EXTINCTION COEFFICIENT CAN BE HELPFUL TO ASSESS THE STOICHI- OMETRY OF THE FLAVIN AND PROTEIN MOIETY IN GENERAL ONE FLAVIN PER ENZYME ACTIVE SITE IS FOUND AND SUBSTOICHIOMETRIC CONCENTRATIONS OF FLAVIN MAY INDICATE DEPLE- TION OF THE FLAVIN DURING PURIFICATION AND HANDLING IN THESE CASES IT MAY BE POSSIBLE TO RECONSTITUTE THE FLAVOPROTEIN BY SUPPLEMENTING THE FLAVIN COFACTOR (SEE CHAPTER 11) THE ABSORBANCE IN THE UV-VISIBLE SPECTRAL RANGE PRESENTS SEVERAL CONVE- NIENT ADVANTAGES: 1 SINCE THE FLAVIN CHROMOPHORE IS AN ESSENTIAL COMPONENT OF THE CATALYTIC MACHIN- ERY, OBSERVABLE SPECTRAL CHANGES DURING THE ENZYMATIC REACTION YIELD IMPORTANT INFORMATION CONCERNING THE REACTION MECHANISM THESE SPECTRAL CHANGES CAN BE OBSERVED USING RAPID REACTION KINETIC INSTRUMENTS (SEE CHAPTER 5) 2 BINDING OF SUBSTRATES, SUBSTRATE ANALOGS AND INHIBITORS CAN BE MONITORED IN MANY CASES BY SPECTROPHOTOMETRY TITRATIONS WITH THE COMPOUND OF INTEREST YIELD DISSOCIATION CONSTANTS AND INFORMATION CONCERNING CHANGES IN THE ACTIVE SITE ENVIRONMENT 3 THE PROTEIN MOIETY PERTURBS THE FLAVIN SPECTRUM YIELDING INFORMATION ON THE FLA- VIN ENVIRONMENT IN THE BINDING POCKET AS WELL AS THE IONIZATION STATE OF THE FLAVIN 4 DURING PROTEIN PURIFICATION, THE FLAVIN ABSORBANCE CAN BE UTILIZED TO LOCATE THE FRACTION OF INTEREST (IN ADDITION TO OTHER PARAMETERS, SUCH AS ACTIVITY, IF KNOWN) THIS IS PARTICULARLY USEFUL WHEN FLAVOPROTEINS ARE PREPARED FROM AN OVERPRODUC- ING HOST SYSTEM ALTHOUGH SOME FLAVOPROTEINS HAVE QUITE DISTINCTIVE UV-VISIBLE SPECTRA IT IS NOT FEASIBLE TO IDENTIFY A FLAVOPROTEIN BY THE SPECTRAL PROPERTIES OF ITS FLAVIN COFAC- TOR ON THE OTHER HAND, THE COURSE OF REDUCTION OF THE OXIDIZED FLAVIN VERY MUCH DEPENDS ON THE PROTEIN MOIETY: THE FLAVIN IS EITHER REDUCED DIRECTLY TO ITS TWO- ELECTRON REDUCED FORM (“HYDROQUINONE”) OR VIA ITS ONE ELECTRON REDUCED STATE (“SEMIQUINONE”) IN THE LATTER CASE, THE FLAVIN SEMIQUINONE MAY EITHER BE PROTO- NATED (NEUTRAL OR BLUE SEMIQUINONE) OR MONODEPROTONATED (ANIONIC OR RED SEMI- QUINONE) THESE TWO RADICAL SPECIES ARE EASILY DISTINGUISHABLE BY THEIR SPECTRAL PROPERTIES: THE NEUTRAL SEMIQUINONE RADICAL HAS A CHARACTERISTIC LONG WAVELENGTH ABSORBANCE AT 580–620 NM (? ?4,000 M–1 CM–1) WHEREAS THE ANIONIC SEMI- QUINONE RADICAL HAS A STRONG ABSORBANCE AROUND 380 NM (? ?16,000 M–1 CM–1) WITH AN ADDITIONAL SHARPER PEAK AT 400 NM (5,6) IT WAS ALSO RECOGNIZED THAT THE PROPENSITY TO STABILIZE THE FLAVIN RADICAL IS LINKED TO THE ENZYMATIC FUNCTION OF THE FLAVOPROTEIN THIS FEATURE WAS EMPLOYED TO CLASSIFY THE LARGE NUMBER OF FLAVOPROTEINS IN AN ATTEMPT TO CORRELATE THE FUNCTION OF THE PROTEIN WITH THE PROPERTIES OF ITS FLAVIN COFACTOR (6,7) THE OCCURRENCE OF THE FLAVIN RADICAL DOES NOT ONLY DEPEND ON THE PROTEIN MOIETY BUT IS ALSO AFFECTED BY OTHER EXTERNAL PHYSICO-CHEMICAL FACTORS, SUCH AS PH EQUALLY IMPORTANT IS THE EFFECT OF LIGANDS FLAVOPROTEIN PROTOCOLS (SUBSTRATE, SUBSTRATE ANALOGS OR INHIBITORS) ON THE GENERATION OF A FLAVIN RADICAL SPECIES THEREFORE, THE REDUCTION OF A GIVEN AND PERHAPS UNKNOWN FLAVOPROTEIN UNDER VARIOUS CONDITIONS YIELDS IMPORTANT BASIC INFORMATION ON THE TYPE AND POSSIBLE REACTION MODE OF THE FLAVOPROTEIN SUBHEADING 2 2, METHOD 2 PROVIDES INFORMATION ON THE REDUCING BEHAVIOR OF A FLAVOPROTEIN REDUCTION OF THE FLAVIN TO ITS TWO-ELECTRON REDUCED FORM RESULTS IN THE LOSS OF THE TWO MAJOR ABSORBANCE PEAKS IN THE UV-VISIBLE RANGE AND CAUSES BLEACHING OF THE FLAVIN CHROMOPHORE (SEE FIG 2) THE PROCEDURE DESCRIBED IN METHOD 2 LEADS TO A STEPWISE REDUCTION OF THE FLAVIN AND HENCE THE ABSORBANCE PEAKS WILL DIMINISH UNTIL COMPLETE REDUCTION IS ACHIEVED WITH THE FINAL SPECTRUM RESEMBLING THAT SHOWN IN FIG 2 FOR REDUCED FMN (FMNRED) ANOTHER NOTABLE FEATURE OF SUCH A STRAIGHTFORWARD REDUCTION TO THE TWO-ELECTRON REDUCED FLAVIN IS AN ISOSBESTIC POINT AROUND 330 NM HOWEVER, IF A FLAVIN SEMI- QUINONE IS STABILIZED BY THE PROTEIN MOIETY THEN REDUCTION WILL RESULT IN THE APPEARANCE OF A FLAVIN RADICAL IDENTIFIABLE BY THE SPECTRAL CHARACTERISTICS MEN- TIONED ABOVE (5,8) CONSEQUENTLY, THE ISOSBESTIC POINTS WILL CHANGE DURING THE COURSE OF THE PHOTOREDUCTION AND WILL DEPEND ON THE RELATIVE CONCENTRATIONS OF OXIDIZED, RADICAL AND REDUCED FLAVIN WHICH ARE DETERMINED BY THE EQUILIBRIUM CONSTANTS OF THE THREE REDOX STATES THE EXTENT OF SEMIQUINONE STABILIZATION CAN BE ESTIMATED BY PLOTTING THE ABSORBANCE AT A WAVELENGTH SOLELY DUE TO THE SEMI- QUINONE SPECIES (E G , THE LONG WAVELENGTH ABSORBANCE OF THE NEUTRAL SEMI- QUINONE ABOVE 580 NM) VS THE TIME OF LIGHT IRRADIATION (OR EQUIVALENTS OF ANOTHER REDUCTANT ADDED) (9) THE METHOD DESCRIBED HERE FOR REDUCTION OF A FLAVOPROTEIN ENJOYS A LONG, SUCCESSFUL HISTORY AND SERVES AS AN IDEAL STARTING POINT (8) FURTHER EXPLORATIONS INTO THE REDUCING PROPERTIES OF A FLAVOPROTEIN CAN BE ACHIEVED WITH SLIGHT ALTERATIONS OF THE PROCEDURE (SEE NOTE 4 ) AND WITH THE SAME EQUIPMENT (SEE SUBHEADING 2 2 ) 2 MATERIALS 2 1 METHOD 1 DETERMINATION OF THE FLAVIN EXTINCTION COEFFICIENT 1 SPECTROPHOTOMETER 2 A MATCHED PAIR OF QUARTZ CUVETS (1-CM PATH LENGTH, SEMIMICRO: 1 2 ML MAX VOL ) 3 FLAVOPROTEIN IN A SUITABLE BUFFER (TRANSPARENT IN THE 300–800 NM RANGE) 4 TRICHLOROACETIC ACID (50%, V/V) 5 SODIUM CARBONATE (SOLID) FOR THE ALTERNATIVE METHODS DISCUSSED IN SUBHEADING 3 1 1 THE FOLLOWING REAGENTS ARE REQUIRED: 6 8 M UREA OR GUANIDINIUM HYDROCHLORIDE 7 SODIUM DODECYLSULFATE (2% W,V) FLAVOPROTEIN PROTOCOLS (DEPENDING ON THE EXTINCTION COEFFICIENT) 2 TRANSFER SAMPLE TO A SEMIMICRO CUVET AND RECORD AN UV-VISIBLE ABSORBANCE SPEC- TRUM FROM 650–300 NM READ ABSORBANCE AT THE MAXIMUM ? 450 (READING1) 3 TRANSFER SAMPLE TO AN EPPENDORF TUBE, ADD 100 µL OF 50% TRICHLOROACETIC ACID (TCA), SPIN DOWN WHITE PRECIPITATE IN A MICROCENTRIFUGE AT MAXIMAL SPEED FOR 2 MIN (SEE NOTE 2) 4 ADJUST PH TO THE ORIGINAL PH USING SOLID SODIUM CARBONATE 5 TRANSFER SUPERNATANT (YELLOW) TO SEMIMICRO CUVET AND RECORD UV/VISIBLE ABSOR- BANCE SPECTRUM FROM 650–300 NM CORRECT THE OBTAINED SPECTRUM FOR THE DILUTION FACTOR READ ABSORBANCE AT THE ABSORBANCE MAXIMUM AT ?450 NM (READING 2) THE EXTINCTION COEFFICIENT OF THE FLAVOPROTEIN CAN NOW BE CALCULATED USING THE FOLLOWING RELATIONSHIP: ?UNKNOWN = ?FMN/FAD × READING 1/READING 3 2 METHOD 2 PHOTOCHEMICAL REDUCTION OF FLAVOPROTEINS 1 DISSOLVE 0 5–1 MG OF THE FLAVOPROTEIN IN 1 ML OF AN APPROPRIATE BUFFER INCLUDING 1 MM EDTA OR POTASSIUM OXALATE AND TRANSFER TO A CUVET RECORD SPECTRUM FROM 800–300 NM FLAVOPROTEIN PROTOCOLS 2 REMOVE OXYGEN WITH THE AID OF EITHER A GAS EXCHANGE LINE OR IN A GLOVE BOX (FOR A BRIEF DESCRIPTION SEE SUBHEADING 2 2 ) TAKE CARE TO KEEP THE CUVET SEALED UNTIL (STEP 5) 3 RECORD ABSORBANCE SPECTRUM IN THE SAME RANGE AS IN (STEP 1) (USING THE OVERLAY OPTION OF THE SPECTROPHOTOMETER SOFTWARE) 4 START REDUCTION BY LIGHT IRRADIATION INITIALLY, SHORT INTERVALS OF 5–10 S ARE RECOM- MENDED RECORD SPECTRUM AFTER EACH REDUCTION STEP UNTIL NO FURTHER SPECTRAL CHANGES ARE OBSERVED 5 OPEN CUVET TO AIR AND RECORD SPECTRUM AFTER COMPLETE REOXIDATION OF THE SAMPLE 4 NOTES 1 IN SOME CASES THE FLAVIN IS COVALENTLY LINKED TO THE PROTEIN MOIETY AND THEREFORE COPRECIPITATES IN THE DENATURATION STEP HOWEVER, COPRECIPITATION WAS ALSO REPORTED FOR OTHER CASES WHERE THE FLAVIN IS NOT COVALENTLY ATTACHED TO THE PROTEIN UNFOLDING OF THE PROTEIN IN 8 M UREA OR GUANIDINIUM HYDROCHLORIDE AND SUBSE- QUENT PRECIPITATION OF THE PROTEIN CAN BE HELPFUL TO DISTINGUISH THESE TWO POSSI- BILITIES (10) 2 ALTHOUGH PRECIPITATION OF THE PROTEIN IN 5% TCA WORKS WELL IN GENERAL, THE LOW PH MAY ALSO CAUSE DECOMPOSITION OF THE FLAVIN THEREFORE THE TIME AT LOW PH SHOULD BE KEPT TO A MINIMUM (SEE STEPS 2–4) IN SOME CASES COOLING TO 4°C HELPS TO PREVENT SUCH UNWANTED DECOMPOSITION IN ORDER TO CONFIRM THE OBTAINED RESULT, TCA CAN BE REPLACED BY ANOTHER DENATURANT, PREFERABLY SODIUM DODECYL SULFATE (USE 0 2% FINAL CONCENTRATION) 3 THE SPECTRUM RECORDED IN SUBHEADING 3 2 , STEP 3 SHOULD BE IDENTICAL TO THE ONE RECORDED INITIALLY CHANGES MAY BE OBSERVED IF THE CONCENTRATION OF THE FLAVOPRO- TEIN IS SLIGHTLY INCREASED DUE TO PARTIAL EVAPORATION OF THE SOLVENT COOLING THE SAMPLE WITH ICE DURING ANEROBIZATION MAY HELP TO PREVENT THIS ALSO, DENATUR- ATION OF THE PROTEIN MAY OCCUR DURING THIS PROCESS (TURBIDITY) THIS CAN BE SPOTTED AT LONGER WAVELENGTH AS A BASELINE DRIFT 4 ANY WHITE LIGHT SOURCE CAN BE USED FOR IRRADIATION (SEE SUBHEADING 2 ) FOR LONGER IRRADIATION TIMES (>1 MIN), IT IS RECOMMENDED THAT THE SAMPLE IS SHIELDED FROM THE DEVELOPING HEAT OF THE LIGHT SOURCE AND THAT THE SAMPLE BE PROPERLY THERMO- STATTED TO THE TEMPERATURE SET IN THE SPECTROPHOTOMETER 5 ALTHOUGH REDUCTION WITH LIGHT IS VERY CONVENIENT FOR MOST APPLICATIONS, A CHEMI- CAL REDUCING AGENT SUCH AS AN ANAEROBIC SOLUTION OF DITHIONITE OR AN ENZYMATIC SYSTEM CAN BE USED TO REDUCE THE FLAVOPROTEIN LIKEWISE, WHERE APPROPRIATE, A REDUCING SUBSTRATE OF THE FLAVOENZYME (OXIDASES, DEHYDROGENASES) CAN BE USED TO ACHIEVE REDUCTION 6 IN SOME CASES PHOTOREDUCTION IS SLOW AND THE USE OF µM CONCENTRATIONS OF 5-DEAZARIBO- FLAVIN AS A CATALYST IS RECOMMENDED (11,12) 7 ESPECIALLY WHEN THE ANIONIC SEMIQUINONE IS ENCOUNTERED, FURTHER PHOTOREDUCTION TO THE FULLY REDUCED FLAVOPROTEIN IS SOMETIMES DIFFICULT TO ACHIEVE THIS SITUATION CAN BE CIRCUMVENTED BY USING AN ALTERNATIVE REDUCTANT (SEE ABOVE) FLAVOPROTEIN PROTOCOLS REFERENCES 1 MASSEY, V AND GHISLA, S (1983) IN BIOLOGICAL OXIDATIONS VOL 34, PP 114–139, SPRINGER-VERLAG, MOSBACH, GERMANY 2 GHISLA, S AND MASSEY, V (1989) MECHANISMS OF FLAVOPROTEIN-CATALYZED REAC- TIONS EUR J BIOCHEM 181, 1–17 3 GHISLA, S , MASSEY, V , LHOSTE, J -M , AND MAYHEW, S G (1974) FLUORESCENCE AND OPTICAL CHARACTERISTICS OF REDUCED FLAVINES AND FLAVOPROTEINS BIOCHEMISTRY 13(3), 589–597 4 MACHEROUX, P , MASSEY, V , THIELE, D J , AND VOLOKITA, M (1991) EXPRESSION OF SPINACH GLYCOLATE OXIDASE IN SACCHAROMYCES CERVISIAE: PURIFICATION AND CHARAC- TERIZATION BIOCHEMISTRY 30, 4612–4619 5 MÜLLER, F , BRÜSTLEIN, M , HEMMERICH, P , MASSEY, V , AND WALKER, W H (1972) LIGHT-ABSORPTION STUDIES ON NEUTRAL FLAVIN RADICALS EUR J BIOCHEM 25(3), 573–580 6 MASSEY, V AND HEMMERICH, P (1980) ACTIVE-SITE PROBES OF FLAVOPROTEINS BIOCHEM SOC TRANS 8, 246–257 7 GHISLA, S AND MASSEY, V (1986) NEW FLAVINS FOR OLD: ARTIFICIAL FLAVINS AS ACTIVE SITE PROBES OF FLAVOPROTEINS BIOCHEM J 239, 1–12 8 MASSEY, V AND PALMER, G H (1966) ON THE EXISTENCE OF SPECTRALLY DISTINCT CLASSES OF FLAVOPROTEIN SEMIQUINONES A NEW METHOD FOR THE QUANTITATIVE PRODUCTION OF FLAVOPROTEIN SEMIQUINONES BIOCHEMISTRY 5, 3181–3189 9 MAYHEW, S G (1971) STUDIES ON FLAVIN BINDING IN FLAVODOXINS BIOCHIM BIOPHYS ACTA 235, 289–302 10 MACHEROUX, P , HILL, S , AUSTIN, S , EYDMANN, T , JONES, T , KIM, S -O , POOLE, R , AND DIXON, R (1998) ELECTRON DONATION TO THE FLAVOPROTEIN NIFL, A REDOX-SENSING TRANSCRIPTIONAL REGULATOR BIOCHEM J , 332, 413–419 11 MASSEY, V AND HEMMERICH, P (1977) A PHOTOCHEMICAL PROCEDURE FOR REDUCTION OF OXIDATION-REDUCTION PROTEINS EMPLOYING DEAZARIBOFLAVIN AS CATALYST J BIOL CHEM 252(16), 5612–5614 12 MASSEY, V AND HEMMERICH, P (1978) PHOTOREDUCTION OF FLAVOPROTEINS AND OTHER BIOLOGICAL COMPOUNDS CATALYZED BY DEAZAFLAVINS BIOCHEMISTRY 17(9), 9–17 FLAVOPROTEIN PROTOCOLS