1 The reaction mechanisms of Groups A and B ﬂ avoprotein monooxygenases ...... 1 1.1 Introduction ...... 1 1.2 Enzymes acting upon aromatic substrates – Group A...... 2 1.2.1 Reactions catalyzed ...... 2 1.2.2 Protein structures ...... 3 1.2.3 Detailed mechanism of PHBH ...... 4 1.2.3.1 Reductive half-reaction ...... 6 1.2.3.2 Oxidative half-reaction ...... 9 1.2.3.3 Hydroxylation chemistry ...... 15 1.2.3.4 Summary ...... 16 1.3 Enzymes acting upon non-aromatic substrates – Group B ...... 16 1.3.1 Reactions catalyzed and subclasses ...... 16 1.3.1.1 BVMOs ...... 17 1.3.1.2 FMOs ...... 19 1.3.1.3 NMOs ...... 20 1.3.1.4 YUCCAs ...... 21 1.3.2 Structural features ...... 22 1.4 References...... 23

2 Flavin-dependent monooxygenases in siderophore biosynthesis ...... 29 2.1 Iron, an essential but scarce nutrient ...... 29 2.2 Siderophores ...... 30 2.2.1 Siderophores are important virulence factors ...... 30 2.2.2 Structural diversity of siderophores ...... 33 2.3 Flavin-dependent N-hydroxylating monooxygenases...... 33 2.4 Catalytic cycle of NMOs ...... 34 2.4.1 Flavin reduction in NMOs ...... 35 2.4.2 Flavin oxidation in NMOs ...... 37 2.5 Three-dimensional structure of NMOs...... 37 2.5.1 FAD-binding domain ...... 40 2.5.2 NADPH-binding domain ...... 40 2.5.3 L-Ornithine-binding domain ...... 41 2.6 The structural basis of substrate speciﬁ city in NMOs ...... 42 2.7 Mechanism of stabilization of the C4a-hydroperoxyﬂ avin by NADP+ ... 42 2.8 Activation of NMOs by amino acid binding ...... 44 2.9 Unusual NMOs ...... 44 2.10 High-throughput screening assay to identify inhibitors of NMOs ...... 45 2.11 Conclusions ...... 46 2.12 References...... 47 x Table of contents

3 The fl avin monooxygenases ...... 51 3.1 Introduction ...... 51 3.2 Occurrence and classifi cation ...... 54 3.2.1 Amino acid sequence motifs ...... 54 3.2.2 DNA screening ...... 55 3.3 Single-component fl avin monooxygenases ...... 56 3.3.1 Subclass A ...... 56 3.3.2 Subclass B ...... 58 3.3.3 Subclass C ...... 60 3.3.4 Subclass D ...... 61 3.3.5 Subclass E ...... 62 3.3.6 Subclass F ...... 62 3.4 Conclusions ...... 64 3.5 References...... 65

4 Structure and catalytic mechanism of NADPH-cytochrome P450 oxidoreductase: a prototype of the difl avin oxidoreductase family of enzymes ...... 73 4.1 Introduction ...... 73 4.2 Properties of CYPOR fl avins ...... 75 4.3 Domain structure and function ...... 78 4.4 Membrane binding domain (MBD) ...... 78 4.5 FMN domain ...... 79 4.6 Cytochrome P450 binding: role of the FMN domain and connecting domain ...... 80 4.7 FAD domain...... 82 4.8 Mechanism of hydride transfer ...... 82 4.9 Interfl avin electron transfer ...... 83 4.10 FMN to heme electron transfer ...... 85 4.11 P450 catalysis ...... 85 4.12 Other CYPOR electron acceptors ...... 87 4.13 CYPOR domain movement and control of electron transfer ...... 87 4.14 Physiological functions of CYPOR and effects of CYPOR defi ciency ... 90 4.15 Human CYPOR defi ciency (PORD) ...... 91 4.16 Contribution of CYPOR to inter-individual variation in human drug metabolism ...... 92 4.17 Unanswered questions and future directions ...... 92 4.18 References ...... 93

5 The xanthine oxidoreductase enzyme family: xanthine dehydrogenase, xanthine oxidase, and aldehyde oxidase ...... 103 5.1 Introduction ...... 103 5.2 Overall structures ...... 104 5.3 Reaction mechanism ...... 105 5.4 Electron transfer from the molybdenum center to other redox-active centers ...... 113 5.5 Reaction of FAD with NAD+ or molecular oxygen ...... 114 Table of contents xi

5.6 Inhibitors of xanthine oxidoreductase ...... 116 5.7 References...... 120

6 Assimilatory nitrate reductase ...... 125 6.1 Introduction and scope...... 125 6.2 Enzyme structure ...... 126 6.3 Kinetics and mechanism...... 131 6.4 Post-translational regulation ...... 133 6.5 Interconversion of sulﬁ te oxidase and nitrate reductase activities ...... 135 6.6 Conclusions ...... 137 6.7 References...... 138

7 Succinate dehydrogenase (Complex II) and fumarate reductase ...... 141 7.1 History of Complex II ...... 141 7.2 Overview of Complex II ...... 143 7.3 Structure of Complex II...... 144 7.4 Catalytic assays ...... 146 7.5 Catalytic mechanism and domain movement ...... 148 7.6 Electron transfer ...... 151 7.7 Quinone-binding site of Complex II ...... 153 7.8 Assembly of the covalent FAD cofactor into Complex II ...... 155 7.9 Concluding remarks ...... 159 7.10 References ...... 159

8 Flavoprotein disulfi de reductases and structurally related fl avoprotein thiol/disulfi de-linked oxidoreductases ...... 165 8.1 Introduction ...... 165 8.2 Group 1 FDR enzymes: classic dithiol/disulfi de oxidoreductases with a single CXXXXC disulfi de redox center...... 169 8.2.1 Dihydrolipoamide dehydrogenase (LipDH) ...... 174 8.2.2 Glutathione reductase (GR) – two new structural studies on this classic member of the group ...... 176 8.2.3 Trypanothione reductase (TryR) ...... 177 8.2.4 Mycothione reductase (MycR) ...... 177 8.3 Group 2A FDR enzymes – enzymes of the Group 1 structural fold requiring an additional C-terminal Cys-based redox center ...... 177 8.3.1 Mercuric ion reductase (MerA) ...... 180

8.3.2 High Mr thioredoxin reductases (TrxR and TGR) ...... 181

8.4 Group 2B FDR enzymes – low Mr thioredoxin reductase (TrxR) and structurally related enzymes ...... 183 8.5 Group 3 FDR enzymes – enzymes with cysteine sulfenic acid or mixed Cys-S-S-CoA redox center ...... 188 8.6 Group 4 FDR enzymes – Group 1-fold enzymes catalyzing novel reactions ...... 192 8.7 Group 5 FDR enzymes – enzymes with a si side pair of Cys residues widely separated in sequence ...... 194 8.8 References...... 196 xii Table of contents

9 Flavoenzymes in pyrimidine metabolism ...... 203 9.1 Introduction ...... 203 9.2 Pyrimidine/dihydropyrimidine interconversions ...... 204 9.2.1 Overview ...... 204 9.2.2 Dihydroorotate dehydrogenases ...... 206 9.2.2.1 General ...... 206 9.2.2.2 Mechanisms of the pyrimidine half-reactions ...... 207 9.2.2.3 Mechanisms of the non-pyrimidine half-reactions ..... 210 9.2.3 Dihydrouridine synthases ...... 213 9.2.3.1 General ...... 213 9.2.3.2 Pyrimidine half-reactions ...... 214 9.2.3.3 Non-pyrimidine half-reaction ...... 214 9.2.4 Dihydropyrimidine dehydrogenases ...... 214 9.2.4.1 General ...... 214 9.2.4.2 Pyrimidine half-reaction ...... 215 9.3 Methylations ...... 215 9.3.1 Overview ...... 215 9.3.2 Flavin-dependent thymidylate synthase ...... 216 9.3.3 Folate/FAD-dependent methyl transferase (TrmFO) ...... 218 9.4 References ...... 221

10 Excited state electronic structure of fl avins and fl avoproteins from theory and experiment ...... 225 10.1 Introduction ...... 225 10.2 Flavin photophysics and the electronic structure of its excited states ...... 227 10.2.1 Moments of the charge distribution ...... 227 10.2.2 Experimental techniques for the determination of excited state electronic structure...... 228 10.3 Linear dichroism measurements of reduced anionic fl avin transition dipole moments and complimentary calculations ...... 229 10.4 Experimental studies of excited state electronic structure of fl avins and complementary calculations ...... 230 10.4.1 Oxidized fl avin ...... 230 10.4.2 Excited state structure of OYE and OYE charge transfer complex ...... 232 ___› Δ 10.4.3 DNA photolyase and µ k0 ...... 234 10.4.4 Experimental results for the fl avin neutral radical...... 236 10.5 Computational studies on fl avins ...... 238 10.5.1 Calculations for oxidized fl avins ...... 238 10.5.2 Computational results for semiquinone fl avin ...... 240 10.5.3 Computational studies on reduced fl avins ...... 241 10.6 Spectroscopic studies bearing on excited electronic states of fl avins 243 10.6.1 Time-resolved studies of oxidized fl avin ...... 243 10.6.2 The triplet state of fl avins ...... 244 10.7 Photoinduced electron transfer in fl avins ...... 244 10.8 Applications of fl avin photochemistry ...... 245 Table of contents xiii

10.9 Conclusions ...... 246 10.10 References ...... 246

11 Structural properties of the alkanesulfonate monooxygenase system that dictate function ...... 255 11.1 Introduction ...... 255 11.2 Sulfur limitation in bacterial systems ...... 256 11.3 FMN reductase of the alkanesulfonate monooxygenase system ...... 258 11.4 Monooxygenase enzymes of the bacterial luciferase family ...... 259 11.4.1 Structural properties of the bacterial luciferase family ...... 259 11.4.2 Structural dynamics of alkanesulfonate monooxygenase ...... 261 11.4.3 Active site structure in the bacterial luciferase family ...... 263 11.4.4 Catalytic mechanisms of the bacterial luciferase family ...... 265 11.4.5 Mechanistic properties of alkanesulfonate monooxygenase .. 265 11.5 Mechanism of ﬂ avin transfer ...... 269 11.6 Conclusions ...... 271 11.7 References ...... 271

12 Single molecule methods to study fl avoproteins ...... 277 12.1 Flavoproteins and electron-transfer reactions ...... 277 12.2 Bulk vs. single-molecule methods ...... 278 12.3 Single-molecule techniques for the study of biological systems ...... 279 12.3.1 Atomic force microscopy ...... 280 12.3.2 Optical tweezers...... 282 12.3.3 AFM based force spectroscopy ...... 284 12.3.3.1 The avidin-biotin complex ...... 286 12.3.3.2 Antigen-antibody interaction ...... 287 12.3.3.3 Molecular interactions in transient complexes...... 287 12.4 Single-molecule experiments with fl avoenzymes ...... 288 12.4.1 Fluorescence measurements ...... 288 12.4.2 Force measurements in fl avoproteins ...... 290 12.5 References ...... 295

13 Applications of Saccharomyces pastorianus Old Yellow Enzyme to asymmetric alkene reductions ...... 299 13.1 Identiﬁ cation and initial characterization ...... 299 13.1.1 History of OYE1 ...... 299 13.1.2 OYE 1 structure and roles of key residues ...... 301 13.1.2.1 Histidine 191 and Asparagine 194 ...... 302 13.1.2.2 Tyrosine 196 ...... 302 13.1.2.3 Threonine 37 ...... 302 13.1.2.4 Tryptophan 116 ...... 304 13.2 Substrate speciﬁ city of OYE 1 ...... 305 13.2.1 Ketones and aldehydes ...... 306 13.2.2 Esters ...... 312 13.2.3 Nitro alkenes ...... 315 13.3 Conclusions ...... 317 13.4 References ...... 318 xiv Table of contents

14 Contributions of protein environment to the reduction potentials of ﬂ avin-containing proteins ...... 321 14.1 Introduction ...... 321

14.2 Computation of Esq/hq on the basis of the crystal structures ...... 322

14.3 Calculation of Esq/hq and determination of redox-linked amino acid residues ...... 324

14.4 Inﬂ uence of the protein backbone conformation on Esq/hq ...... 325

14.5 Inﬂ uence of the loop region near the ﬂ avin binding site on Esq/hq ...... 326

14.6 Inﬂ uence of the FMN phosphate group on Esq/hq...... 329 14.7 Conclusions ...... 332 14.8 References ...... 332

15 Methods based on continuum electrostatics and their application to fl avoproteins – a review ...... 335 15.1 Introduction ...... 335 15.2 The continuum electrostatic model based on the Poisson-Boltzmann equation ...... 336 15.2.1 The physical basis of the Poisson-Boltzmann equation ...... 336 15.2.2 Electrostatic potentials and electrostatic energies ...... 339 15.3 Association of fl avoproteins ...... 341 15.3.1 Electrostatic docking of fl avoproteins ...... 341 15.3.2 Similarity of electrostatic potentials of proteins ...... 342 15.4 Titration behavior of proteins ...... 344 15.4.1 Microstate model ...... 345 15.4.2 DTPA – An illustrative example ...... 346 15.4.3 Theoretical analysis of the protonation of fl avoproteins ...... 349 15.5 Recent and upcoming developments ...... 352 15.6 References ...... 354

16 Flavoproteins and blue light reception in plants ...... 361 16.1 Introduction (light reception in plants) ...... 361 16.2 Plant phototropins ...... 363 16.2.1 LOV domain structure ...... 364 16.2.2 LOV photochemistry ...... 365 16.2.3 LOV signal propagation ...... 369 16.3 Cryptochromes ...... 371 16.3.1 Cryptochrome structure ...... 371 16.3.2 Cryptochrome photochemistry ...... 374 16.3.3 Cryptochrome signal transduction ...... 378 16.4 Outlook ...... 380 16.5 References ...... 381

17 Ultrafast dynamics of fl avins and fl avoproteins ...... 393 17.1 Introduction ...... 393 17.2 Ultrafast dynamics of fl avins ...... 394 17.2.1 Steady-state spectroscopic properties ...... 394 17.2.2 Oxidized fl avins ...... 396 Table of contents xv

17.2.3 Anionic and neutral radical fl avins ...... 398 17.2.4 Anionic and neutral fully-reduced fl avins ...... 399 17.3 Electron transfer in model fl avodoxin ...... 401 17.3.1 Experiment design, reaction scheme and probing strategy ..... 401 17.3.2 Femtosecond charge separation, frozen active-site confi guration and critical free energies ...... 403 17.3.3 Ultrafast charge recombination, vibrational quantum effect and hot ground-state cooling ...... 404 17.3.4 Photoinduced redox cycle, reaction time scales, and vibrational coupling generality ...... 406 17.4 Enzymatic reactions and repair photocycles in DNA photolyases ...... 408 17.4.1 Dynamics and mechanism of cyclobutane pyrimidine dimer repair by CPD photolyase ...... 408 17.4.1.1 Sequential splitting dynamics of the cyclobutane ring ...... 408 17.4.1.2 Electron tunneling pathways and functional role of adenine moiety ...... 410 17.4.2 Dynamics and mechanism of repair of UV-induced (6-4) photoproduct by (6-4) photolyase ...... 414 17.4.2.1 Ultrafast electron and proton transfer dynamics ...... 416 17.4.2.2 Catalytic repair photocycle ...... 417 17.5 Signal transduction in blue-light photoreceptors ...... 419 17.5.1 Photoaddition of cysteine to fl avin in phototropin ...... 419 17.5.2 Switching of fl avin hydrogen bond in BLUF protein ...... 419 17.5.3 Ultrafast fl avin dynamics in cryptochrome ...... 420 17.6 Conclusions ...... 420 17.7 References ...... 421

Index ...... 429