Index

Absorbance. see Ultraviolet-visiblespectroscopy

ADR, see Adrenodoxin reductaseAdrenodoxin reductase (ADR), circular

dichroism analysis, 120, 121Analogs, see Flavin analogs

B

Basis set, see Quantum chemicalcomputations

Bond order, quantum chemicalcomputations, 216

7a-Bromo-acetyl-10-methyl-isoallloxazine,

molecular maquette, concept, 195,196

peptide flavination,flavination reaction and product

isolation, 202-204materials, 197peptide synthesis, 200, 202

synthesis,7-acetyl-10-methyl-isoalloxazine

preparation, 198-200, 204bromination reaction, 200, 204,

2054-chtoro-3-nitro-acetophenone

preparation, 197, 1984-methylamino- 3 -nitro-

acetophenone preparation,198

materials, 196, 197, 204

CD, see Circular dichroism

Charge distribution, quantum chemicalcomputations, 216, 223—225

Circular dichroism (CD),adrenodoxin reductase, 120, 121data presentation and analysis, 115,

116electron transferring fiavoprotein,

119flavocytochrome P-450 BM3, 118,

119guidelines for successful

flavoprotein analysis, 121information available from

flavoprotein experiments, 112—115

instrumentation, 114, 115lipoamide dehydrogenase, 118, 120nitrate reductase, 120sample preparation, 115sensitivity and sample requirements,

116, 117theory, 111, 112

Computation, see Quantum chemicalcomputations

Covalent flavoproteins,catalysis of flavinylation, 181, 182identification of flavoproteins, 183,

1S9linkage analysis,

aminoacyl flavin isolation, 185,189, 190

6-S-cysteinyl flavin absorptionspectra, 186, 187

8a-5-cysteinyl riboilavins,fluorescence analysis, 188,190

247

248 Index

histidine-linked flavinfluorescence spectra, 187,190

mass spectrometry, 186materials, 182, 183peptide isolation, 184, 185, 189standards, 188, 189strategy, 184tyrosine-linked flavin,

absorption spectra, 187fluorescence spectra, 188, 190

linkage types, 181, 182Cysteine-linked flavins, see Covalent

flavoproteins

D

5-Deaza-5-carbafiavins, mechanismstudies of flavoenzymes,166-169

Disulfide reductase, see Lipoamidedehydrogenase

freeze quench studies, see Freezequench

theory, 99, 100Electron transferring flavoprotein

(ETF), circular dichroismanalysis, 119

Erythrocyte glutathione reductaseactivation coefficient (EGRAC),

chemotherapy patients, 237determination,

calculations, 232confounding factors, 235, 236materials, 230principle, 231,232

field studies, 235premature infants, 236

ESR, see Electron spin resonanceETF, see Electron transferring

flavoproteinExtinction coefficient see Ultraviolet-

visible spectroscopy

E

EGRAC, see Erythrocyte glutathionereductase activation coefficient

Electron spin resonance (ESR),flavin semiquinone analysis,

catalysis studies,catalytic competence of flavin

semiquinones, 107, 108semiquinone formation, 107

data acquisition, 101, 102g-factor, J03, 104glucose oxidase semiquinones,

102linewidth, 102, 103overview, 97-99quantitative analysis, 105sample preparation, 100, 101signal saturation, 105, 106

FAD, see Flavin adenine dinucleotideFlavin adenine dinucleotide (FAD),

affinity for flavoproteins, 149analogs in flavoprotein analysis,

advantages over other approaches,157,158

isoalloxazine ring systemaccessibility studies,

pyrazine ring, 162, 163pyrimidine ring, 1608-substituted flavins, 165, 166substitution sites in analogs,

160-162xylene ring, 163, 164

mechanistic probing,5-deaza-5-carbaftavins, 166-

1698-substituted flavins, 168, 169

oxidation-reduction potentials.

Index 249

Hammett s value correlation,172-174

mechanistic implications, 174,175

modification by proteininteractions, 169, 170,173, 174

pyrimidine substitutionsensitivity, 172, 173

values, 170-172rationale, 160

biosynthesis, 1chemical synthesis, 158, 159fluorescence assays,

apoflavodoxin quenching assay,35-37,41

fluorescence properties ofcofactors and fiavoproteins,overview, 26—28

pH-dependent assay, 38, 41,42phosphodiesterase-based assay,

calibration and linearity test,12,22

data acquisition, 12, 13, 22data analysis, 13—16denaturation of protein, 13—15materials, 10, 11principle, 11, 12

fluorescence properties of cofactorand flavoproteins, overview,26-28

forms in catalysis, 207, 208identification and quantification in

flavoproteins,high-performance liquid

chromatography assay,calibration curve construction,

20data analysis, 20, 22principle, 18, 20sample preparation, 20

materials, 10,11overview, 9, 10

ultraviolet-visible spectroscopyassay,

heat denaturation, 17, 22iron-sulfur flavoproteins, 18phosphodiesterase treatment,

17sodium dodecyisulfate

treatment of simpleflavoproteins, 16, 17, 22

redox potential, 49, 50removal from flavoproteins,

overview of techniques, 149semiquinone properties, 97, 98structure, 139, 140, 159

Flavin analogs, see also 7a-Bromo-acetyl-10-methyl-isoallloxazine,

advantages over other flavoproteinanalysis approaches, 157, 158

isoalloxazine ring systemaccessibility studies,

pyrazine ring, 162, 163pyrimidine ring, 1608-substituted flavins, 165, 166substitution sites in analogs. 160—

162xylene ring, 163, 164

mechanistic probing,5-deaza-5-carbaflavins, 166-169^substituted flavins, 168, 169

oxidation-reduction potentials,Hammett s value correlation,

172—174mechanistic implications, 174,

175modification by protein

interactions, 169, 170, 173,174

pyrimidine substitutionsensitivity, 172,173

values, 170-172rationale for flavoprotein analysis,

160Flavin mononucleotide (FMN),

250 Index

affinity for flavoproteins, 149, 150analogs in flavoprotein analysis,

advantages over other approaches,157, 158

rationale,160

isoalloxazine ring systemaccessibility studies,

pyrazine ring, 162, 163pyrimidine ring, 3608-substituted flavins, 165, 166substitution sites in analogs,

160-162xylene ring, 163, 164

mechanistic probing,5-deaza-5-carbaflavins, 166—

1698-substituted flavins, 168, 169

oxidation-reduction potentials,Hammett a value correlation,

172-174mechanistic implications, 174,

175modification by protein

interactions, 169, 170,173,174

pyrimidine substitutionsensitivity, 172, 173

values, 170-172biosynthesis, 1chemical synthesis, 158, 159fluorescence assays,

apoflavodoxin quenching assay,35-41

fluorescence properties ofcofactors and flavoproteins,overview, 26-28

pH-dependent assay, 38, 41,42pb osp hod iesterase-based assay,

calibration and linearity test,12,22

data acquisition, 12, 13, 22data analysis, 13-16

denaturation of protein, 13—15materials, 10,11principle, 11,12

forms in catalysis, 207, 208identification and quantification in

flavoproteins,high-performance liquid

chromatography assay,calibration curve construction,

20data analysis, 20, 22principle, 18, 20sample preparation, 20

materials, 10,11overview, 9,10ultraviolet-visible spectroscopy

assay,heat denaturation, 17,22iron-sulfur flavoproteins, 18phosphodiesterase treatment,

17sodium dodecylsulfate

treatment of simpleflavoproteins, 16, 17,22j

reconstitution in flavoproteins,apoenzyme stability, 152, 153flavin incubation, 152, 154materials, 151yield determination, 152—154

retiox potential, 49, 50removal from flavoproteins,

materials, 150, 151overview of techniques, 149, 150pheny) Sepbarose

chromatography, 151—153semiquinone properties, 97, 98structure, 139, 140, 159

Flavocytochrome b2

dissociation constants for flavinmononucleotide, 150

flavin moTionucleotidereconstitution,

Index 251

apoenzyme stability, 152, 153flavin incubation, 152, 154materials, 151yield determination, 152-154

flavin monoiiucleotide removal,materials, 150, 151overview of techniques, 149, 150phenyl Sepharose

chromatography, 151-153freeze quench studies, 88

Flavocytochrome P-450 BM3circular dichroism analysis, 118, 119Raman spectroscopy, 134, 135

Flavoprotein mono oxygen a ses, see also/5-Hydroxyben^oate hydroxylase,

classification, 67general reaction, 67quantum mechanical calculations of

nudeophilic reactivity andactivation barriers, 220, 221,223

Flavoprotein oxidases, see alsoVanillyl-alcohol oxidase,

general reaction, 63half-reactions, 64, 65rate-limiting step, 63

Fluorescence spectroscopy,cuvets, 29denaturation studies of flavoproteins,

32-34emission spectra, collection for

flavoproteins, 31, 32,41,43excitation spectra, collection for

flavoproteins, 29-31,41, 43,44

flavin cofactor identification andquantification,

apoflavodoxin quenching assay,35-37,41

fluorescence properties ofcofactors and flavoproteins,overview, 26—28

pH-deptmdent assay, 38,41, 42

phosphodiesterase-based assay,calibration and linearity test,

12, 22data acquisition, 12, 13, 22data analysis, 13-16denaturation of protein, 13—15materials, 10, IIprinciple, 11, 12

inner filter effect, 43instrumentation, 28, 29, 44lifetimes, 26, 39,42linkage identification in covalent

flavoproteins,8a-S-cysteinylriboflavins, 188,

190standards, 188, 189tyrosine-linked flavins, 188, 190

polarization of flavin fluorescence,39,42

quantum yield, 26quenching studies of flavoproteins,

39,40, 42reduced flavoproteins, 40, 41renaturation studies of flavoproteins,

32, 34, 35reproducibility, 44temperature sensitivity, 43,44theory, 25, 26

FMN, see Flavin mononueleotideFreeze quench,

aging and spraying into cold solvent,91,92

flavocytochrome b2, 88materials and apparatus, 89-91, 93nonflavin enzymes, 88, 89principles, 87, 88ram control, 90spectroscopic analysis, 87-89stopping the reaction, 92temperature control, 90temperature dependence of electron

transfer reactions,92,93

252 index

g-Factor, see Electron spin resonanceGeometry, optimization with quantum

chemical computations, 216, 218,219

Glucose oxidase, electron spinresonance of flavin semiquinones,102

Glucose-6-phosphate dehydrogenasedeficiency, erythrocyteglutathione reductase activationcoefficient in patients, 235

Glutathione reductase (GR),erythrocyte glutathione reductase

activation coefficientdetermination,

calculations, 232confounding factors, 235, 236materials, 230principle, 231, 232

nitric oxide donor modification,applications, 240crystallization of modified

protein, 234, 239, 240materials, 230physiological significance, 239,

240reaction conditions, 233

reversible denaturation assay fordrug testing,

denaturadon reaction, 234materials, 230, 231peptidomimetic inhibitors, 240,

241rationale, 234, 240renaturation and assay, 234, 235

GR, see Glutathione reductase

H

Hammett s value, oxidation-reductionpotential correlation, 172—174

Heat of formation, quantum chemicalcomputations, 216

High-performance liquidchromatography (HPLC), flavincofactor identification andquantification,

calibration curve construction, 20data analysis, 20, 22materials, 10, 11principle, 18,20sample preparation, 20

Highest occupied molecular orbital(HOMO), quantum chemicalcomputations,

nucleophilic reactivity and activationbarriers in flavoproteinmonooxygenases, 220, 221,223

orbital energy and distribution, 216Histidine-linked flavins, see Covalent

flavoproteinsHOMO, see Highest occupied

molecular orbitalHPLC, see High-performance liquid

chromatography^-Hydroxybenzoate hydroxylase

(PHBH),activity assay and steady-state

kinetics, 68, 70,77,78,81quantum chemical computation,

nucleophilic reactivity andactivation barriers, 220, 221,223

reaction catalyzed, 68substrate specificity, 68transient kinetic studies,

overview, 68-70oxidative half-reaction, 79-81reductive half-reaction, 78, 79, 81

Hypothyroidism, diagnosis witherythrocyte glutathione reductaseactivation coefficient, 236

Index 253

l

Infrared spectroscopy, protein studies,126

Inner filter effect, fluorescence,43

Lipoamide dehydrogenase,absorbance spectroscopy, 63activity assays,

materials, 70, 80overview, 62standard assay, 71steady-state kinetics, 71, 72, 80

circular dichroism analysis, 118, 120reaction catalyzed, 62transient kinetic studies,

NADH reduction of oxidizedenzyme, 73, 74, 80

overview, 62, 63, 74oxidative half-react ion, 73, 80reductive half-reaction, 72, 73, 80

Lowest unoccupied moJecular orbital(LUMO),

oxidation-reduction potential, lineardependence of energy, 172

quantum chemical computations,charge effects, 223, 224nucleophilic reactivity and

activation barriers inflavo proteinmonooxygenases, 220,221,223

orbital energy and distribution,216

redox potential calculation foTsubstituted flavins, 220, 221

LUMO, see Lowest unoccupiedmolecular orbital

M

Molecular maquette,

definition, 195flavin analog for synthesis, see 7a-

Bromo-acety i -10-methyl-isoalloxazine,

flavin binding site design,195, 196

N

Nitrate reductase, circular dichroismanalysis, 120

Nitric oxide (NO), modification ofglutathione reductase,

applications, 240crystallization of modified protein,

234,239, 240materials, 230physiological significance, 239, 240reaction conditions, 233

NMR, see Nuclear magnetic resonanceNO, see Nitric oxideNuciear magnetic resonance (NMR),

instrumentation, 142isotope enrichment, 144one-dimensional experiments, 142,

143paramagnetic effects, 140, 141, 146phosphorous-31 resonance

experiments, 144pTOtoi) resonance experiments, 143,

144sample preparation, 139-142sensitivity and sample requirements,

139, 140, 144shimming/field homogeneity/peak

overlap, 145three-dimensional experiments, 142,

143tubes, 145

OOxidation-reduction potential,

flavin analogs,

254 Index

Hammctt o value correlation.172-174

mechanistic implications,174,175

modification by proteininteractions, 169,170,173,174

pyrimidine substitutionsensitivity, 172, 173

values, 170-172flavin cofactors, 49, 50flavoproteins,

factors affecting redox values, 50,98,99

measurement techniques,overview, 50, 51

spectreiectrochemicalmeasurement,

assembly of apparatus, 57cuvet preparation, 56, 58data acquisition and analysis,

57, 58materials, 52, 54—56overview, 51, 52sodium dithionite preparation,

56, 57lowest unoccupied molecular orbital,

linear dependence, 172reference solutions in measurement,

49,51

PDE, see PhosphodiesterasePeptide flavination, see 7a-Bromo-

acetyl-10-methyl-isoallloxazinePHBH, see p-Hydroxybenzoate

hydro xylasePhosphodiesterase (PDE), assays for

flavin cofactor identification,fluorescence,

11-16ultraviolet-visible spectroscopy, 17,

18

Photochemical reduction, seeUltraviolet-visible spectroscopy

Polarization, see Fluorescencespectroscopy

Presteady-state kinetics, see Freezequench; Stopped-flowspectroscopy

Protein-energy malnutrition, effect onerythrocyte ghitathione reductaseactivation coefficient, 235, 236

Quantum chemical computations,ab initio methods, 209, 210, 213,

214,218,225,226failures and troubleshooting, 217,

218geometry, optimization, 216, 218,

219hardware, 209, 210nucleophilic reactivity and activation

barriers in flavoproteinmonooxygenases, 220, 221,223

redox potential calculation forsubstituted flavins, 220, 221

software, 207-209steps in analysis,

calculation model selectionfactors,

basis sets, 214, 215,226charge of system, 211, 212,

224, 225, 227computation time, 214, 215environmental effects, 212,

213initial geometry, 211quantum mechanical method,

213,214,225,226output parameters, defining and

selection, 215,216

Index 255

running of job, 217starting geometry, defining, 210,

211validation and analysis, 217, 227

R

Raman spectroscopy,applications, 125fluorescence quenching, 135interpretation of flavoprotein

spectra, 132-135resonance Raman spectroscopy,

data acquisition, 128materials, 126, 127sample preparation, 128

semiquinone formation for studies,127,128, 131, 132

surface enhanced resonance Ramanscattering,

coIJoidal silver preparation, 127,129-131,135,136

materials, 127sample preparation, 127, 131

theory, 126Rapid-reaction kinetics, see Freeze

quench; Stopped-flowspectroscopy

Redox potential, see Oxidation-reduction potential

Riboflavin,clinical interventions,deficiency correction, 237

disease treatment and prevention,237, 238

medication compliancemonitoring with high doses,238

precautions with riboflavinsolutions, 233

premature infants, 236tissue glue application, 238

deficiency monitoring, seeErythrocyte glutathionereductase activation coefficient

Riboflavin,redox potential, 49, 50structure, 1, 2, 139, 140,159

s, see Hammett cr valueSemiquinone,

electron spin resonance analysis,catalysis studies,

catalytic competence of flavinsemiquinones, 107, 108

semiquinone formation, 107g-factor, 103, 104glucose oxidase semiquinones,

102linewidth, 102, 103overview, 97-99quantitative analysis, 105sample preparation, 100, 101signal saturation, 105, 106

formation for Raman spectroscopystudies, 127, 128,131, 132

properties, overview, 97, 98ultraviolet-visible spectroscopy, 3,

98SERRS, see Surface enhanced

resonance Raman scatteringSpin distribution, quantum chemical

computations, 216Stopped-flow spectroscopy,

lipoamide dehydrogenase,NADH reduction of oxidized

enzyme, 73, 74, 80overview, 62, 63, 74oxidative half-reaction, 73, 80reductive half-reaction, 72, 73, 80

/j-hydroxybenzoatc hydroxylase,overview, 68—70oxidative half-reaction, 79-81

256 Index

reductive half-reaction, 78, 79, 81vanillyl-alcohol oxidase,

oxidative ha if-reaction, 76, 77, 81reductive half-reaction, 66, 67,

75,76,80,81Surface enhanced resonance Raman

scattering (SERRS), see Ramanspectroscopy

Thioredoxin reductase (TrxR),reversible denaturation assay fordrug testing,

denaturation reaction, 234materials, 230, 231peptidomhnetic inhibitors, 240, 241rationale, 234, 240renaturation and assay, 234, 235

Transient kinetics, see Freeze quench;Stopped-flow spectroscopy

TrxR, see Thioredoxin reductaseTyrosine-iinked flavins, see Covalent

flavoproteins

Ultraviolet-visible spectroscopy, seealso Stopped-flow spectroscopy,

applications with flavoproteins,overview, 3, 4

extinction coefficient, determinationfor flavins,

calculations, 5materials, 4precipitation of protein!! 5, 6

flavin analog accessibility s udies inflavoproteins, 165, 166

flavin cofactor identification ,mdquantification,

heat denaturation, 17, 22iron-suifur flavoproteins, 18materials, 10, 11phosphodiesterase treatment, 17sodium dodecylsulfate treatment

of simple flavoproteins, 16,17,22

flavin semiquinones, 3, 98hydroquinorte versus semiquinone

flavins, 3linkage identification in covalent

flavoproteins,6-5-cysteinyl flavins, 186, 187tyrosine-iinked flavins, 187

oxidized versus reduced flavincofactors, 1,2,4,27,28

photochemical reduction offlavoproteins,

catalysts, 6irradiation of sample, 5, 6materials, 5overview, 4

Vanillyi-alcohol oxidase,assay and steady-state kinetic

analysis, 74, 75rate-limiting step, 64, 65, 67reaction catalyzed, 65structure, 65transient kinetic studies,

oxidative half-reaction, 76,77,81

reductive half-reaction, 66, 67,75,76,80,81

Vibrational spectroscopy, see Infraredspectroscopy;

Raman spectroscopyVinca alkyloids, riboflavin inactivation,

229,233