1. Introduction 148

2. Basics of X-ray and neutron scattering 149

2.1 Elastic scattering of electromagnetic radiation by a single electron 149

2.2 Scattering by assemblies of electrons 151

2.3 Anomalous scattering and long wavelengths 153

2.4 Neutron scattering 153

2.5 Transmission and attenuation 155

3. Small-angle scattering from solutions 156

3.1 Instrumentation 156

3.2 The experimental scattering pattern 157

3.3 Basic scattering functions 159

3.4 Global structural parameters 161

3.4.1 Monodisperse systems 161

3.4.2 Polydisperse systems and mixtures 163

3.5 Characteristic functions 164

4. Modelling 166

4.1 Spherical harmonics 166

4.2 Shannon sampling 169

4.3 Shape determination 170

4.3.1 Modelling with few parameters: molecular envelopes 171

4.3.2 Modelling with many parameters: bead models 173

4.4 Modelling domain structure and missing parts of high-resolution models 178

4.5 Computing scattering patterns from atomic models 184

4.6 Rigid-body refinement 187

5. Applications 190

5.1 Contrast variation studies of ribosomes 190

5.2 Structural changes and catalytic activity of the allosteric enzyme ATCase 191

6. Interactions between molecules in solution 203

6.1 Linearizing the problem for moderate interactions: the second virial coefficient 204

6.2 Determination of the structure factor 205

7. Time-resolved measurements 211

8. Conclusions 215

9. Acknowledgements 216

10. References 216

A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies – on ribosomes and on the allosteric protein aspartate transcarbamoylase – illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.

1. Helicases as components of macromolecular machines 3

2. Helicases in replication 7

2.1 The loading of replicative helicases 7

2.1.1 Loading Rep helicase at the replication origin of bacteriophage ϕX174 7

2.1.2 How is a ssDNA strand passed through (and bound in?) the central channel of the hexameric replicative helicases? 8

2.1.3 Loading of E. coli DnaB helicase in the absence of an auxiliary protein-loading factor 8

2.1.4 The T7 gp4 primase-helicase is loaded by means of a facilitated ring-opening mechanism 10

2.1.5 Bacteriophage T4 gp61 primase can be viewed as a loading factor for the homologous gp41 helicase 11

2.1.6 DnaC serves as the loading factor for E. coli DnaB helicase 11

2.1.7 The role of bacteriophage T4 gp59 in loading the T4 gp41 helicase 12

2.1.8 Loading of helicases onto ssDNA covered by ssDNA-binding proteins (SSBPs) 15

2.2 DNA polymerase and ssDNA-binding proteins can serve as reporters for replicative helicases in their elongation mode 17

2.2.1 The DNA polymerase, the sliding clamp, and the clamp loader 17

2.2.2 The role of ssDNA-binding protein 18

2.2.3 Coupling is achieved by the DNA polymerase and the ssDNA-binding protein 18

2.3 Arrest of replicative helicases 18

2.3.1 The Ter sites and termination proteins 19

2.3.2 Models for orientation-specific fork arrest 20

3. Helicases in transcription 20

3.1 Assisted loading of E. coli RNAP by the sigma70 initiation factor 21

3.1.1 RNAP holoenzyme formation 23

3.1.2 Formation of closed promoter complexes RPc and RPi 24

3.1.3 Strand separation and the formation of the open complex 24

3.1.4 Promoter clearance 24

3.1.5 Conclusions 25

3.2 Transcript formation serves as a monitor (reporter) of RNAP helicase activity in the elongation phase of transcription 25

3.2.1 Structural aspects of transcription complex translocation 26

3.2.2 Transcript elongation is an approximately isoenergetic process 26

3.3 Termination of transcription 27

3.3.1 Intrinsic termination 27

3.3.2 Termination by transcription-termination helicase Rho 28

3.3.3 Conclusions 29

3.4 Loading of the Rho transcription-termination helicase 29

4. Helicases in nucleotide excision repair (NER) 30

4.1 The limited strand-separating activity of the UvrAB complex 31

4.2 UvrB is a DNA helicase adapted for NER 33

4.2.1 The ATP-binding site of UvrB is similar to that of other helicases 33

4.2.2 The putative DNA-binding site 33

4.3 UvrA as a UvrB loader 34

4.4 Assisted targeting of UvrAB to the transcribed strand of DNA sequences undergoing active transcription 34

4.4.1 Targeting of UvrAB to damaged DNA sites in the vicinity of promoters is assisted by RNAP 34

4.4.2 TRCF participates in the assisted targeting of UvrAB to a transcribing RNAP stalled by a DNA lesion 35

4.4.3 Conclusions 36

4.5 UvrC endonuclease is the reporter of UvrAB helicase activity in incision 36

4.6 Post-incision events 36

4.7 Mechanistic details of the helicase activity of UvrD 37

4.7.1 Structural organization and conformational changes 37

4.7.2 Translocation and unwinding activities 38

4.7.3 Step size of DNA unwinding 38

4.7.4 Oligomeric state 39

5. Helicases in recombination 39

5.1 Role of RecBCD and RecQ in the initiation of recombination 40

5.1.1 The RecBCD enzyme 40

5.1.1.1 Loading of RecBCD onto its DNA substrate does not require a separate loading protein 40

5.1.1.2 The endonuclease activity of RecD, and the binding of SSB protein, serve as reporters of RecBCD helicase activity 40

5.1.1.3 RecA can also serve as a reporter of RecBCD helicase activity 41

5.1.1.4 RecBCD step size and unwinding mechanism 41

5.1.1.5 RecBCD unwinding efficiency 42

5.1.2 The RecQ protein 43

5.2 Strand-exchange reaction catalyzed by RecA 43

5.2.1 The nucleoprotein filament 44

5.2.2 The strand-exchange reaction 46

5.2.2.1 A ‘minor-groove’ to ‘major-groove’ triple-helix transition 46

5.2.2.2 Role of the secondary DNA-binding site of RecA 46

5.2.2.3 SSB protein stimulates the strand-exchange reaction 46

5.2.2.4 Cost of the strand-exchange reaction 47

5.2.3 Conclusion: RecA is a ‘scaffolding’ protein that prepares DNA for a coupled unpairing–reannealing reaction 48

5.3 Role of the RuvAB helicase in processing recombination intermediates by a branch migration mechanism 48

5.3.1 A brief description of the RuvA and RuvB proteins 49

5.3.2 Crystal structures of RuvA and the RuvA–Holliday junction complex 50

5.3.3 RuvA as a scaffolding protein that prepares the homoduplex for strand separation 51

5.3.4 Branch migration mechanism 51

6. RNA unwindases in the spliceosome 52

6.1 RNA structural rearrangements within the spliceosome: an overview 52

6.2 The spliceosome consumes chemical free energy 54

6.3 RNA structural alterations require the concerted (or coupled) action of unwinding and reannealing proteins 54

6.4 The reannealing proteins of the spliceosome: contribution of the RNA recognition motifs (RRMs) 55

6.5 The RNA unwindases of the spliceosome 55

6.6 RNA targets of the RNA unwindases 56

7. Conclusions and overview 57

8. Acknowledgments 58

9. References 59

In Part I of this review [Delagoutte & von Hippel, Quarterly Reviews of Biophysics (2002) 35, 431–478] we summarized what is known about the properties, mechanisms, and structures of the various helicases that catalyze the unwinding of double-stranded nucleic acids. Here, in Part II, we consider these helicases as tightly integrated (or coupled) components of the various macromolecular machines within which they operate. The biological processes that are considered explicitly include DNA replication, recombination, and nucleotide excision repair, as well as RNA transcription and splicing. We discuss the activities of the constituent helicases (and their protein partners) in the assembly (or loading) of the relevant complex onto (and into) the specific nucleic acid sites at which the actions of the helicase-containing complexes are to be initiated, the mechanisms by which the helicases (and the complexes) translocate along the nucleic acids in discharging their functions, and the reactions that are used to terminate the translocation of the helicase-containing complexes at specific sites within the nucleic acid ‘substrate’. We emerge with several specific descriptions of how helicases function within the above processes of genetic expression which, we hope, can serve as paradigms for considering how helicases may also be coupled and function within other macromolecular machines.

1. Introduction 258

2. Set-up of MD simulations 260

2.1 Constant-pressure dynamics 260

2.2 Grand-canonical dynamics 261

2.3 Boundary conditions 261

3. Force fields 262

3.1 Proteins 262

3.2 Nucleic acids 265

3.3 Carbohydrates 266

3.4 Phospholipids 266

3.5 Polarization 267

4. Electrostatics 267

4.1 Spherical truncation methods 268

4.2 Ewald summation methods 269

4.3 Fast multipole (FM) methods 271

4.4 Reaction-field methods 271

5. Implicit solvation models 271

6. Speeding-up the simulation 273

6.1 SHAKE and its relatives 273

6.2 Multiple time-step algorithms 274

6.3 Other algorithms 275

7. Conformational space sampling 275

7.1 Multiple copy simultaneous search (MCSS) and locally enhanced sampling (LES) 275

7.2 Steered or targeted MD 276

7.3 Self-guided MD 276

7.4 Leaving the standard 3D Cartesian coordinate system: 4D MD and internal coordinate MD 277

7.5 Temperature variations 277

8. Thermodynamic calculations 278

8.1 Lambda (λ) dynamics 278

8.2 Extracting thermodynamic information from simulations 279

8.3 Non-Boltzmann thermodynamic integration (NBTI) 279

8.4 Other methods 279

9. QM/MM calculations 282

10. MD simulations of protein folding and unfolding 283

10.1 High-temperature effects 284

10.2 Co-solvent and polarization effects 288

10.3 External force effects 288

11. On the horizon 291

12. Acknowledgements 292

13. References 292

Molecular dynamics simulations are widely used today to tackle problems in biochemistry and molecular biology. In the 25 years since the first simulation of a protein computers have become faster by many orders of magnitude, algorithms and force fields have been improved, and simulations can now be applied to very large systems, such as protein–nucleic acid complexes and multimeric proteins in aqueous solution. In this review we give a general background about molecular dynamics simulations, and then focus on some recent technical advances, with applications to biologically relevant problems.

1. Introduction 374

2. Metals in biology 378

3. The targets: structure and function of ion channels 380

4. General effects of metal ions on channels 382

4.1 Three types of general effect 382

4.2 The main regulators 383

5. Effects on gating: mechanisms and models 384

5.1 Screening surface charges (Mechanism A) 387

5.1.1 The classical approach 387

5.1.1.1 Applying the Grahame equation 388

5.1.2 A one-site approach 391

5.2 Binding and electrostatically modifying the voltage sensor (Mechanism B) 391

5.2.1 The classical model 391

5.2.1.1 The classical model as state diagram – introducing basic channel kinetics 392

5.2.2 A one-site approach 395

5.2.2.1 Explaining state-dependent binding – a simple electrostatic mechanism 395

5.2.2.2 The relation between models assuming binding to smeared and to discrete charges 396

5.2.2.3 The special case of Zn2+ – no binding in the open state 396

5.2.2.4 Opposing effects of Cd2+ on hyperpolarization-activated channels 398

5.3 Binding and interacting non-electrostatically with the voltage sensor (Mechanism C) 398

5.3.1 Combining mechanical slowing of opening and closing with electrostatic modification of voltage sensor 400

5.4 Binding to the pore – a special case of one-site binding models (Mechanism D) 400

5.4.1 Voltage-dependent pore-block – adding extra gating 401

5.4.2 Coupling pore block to gating 402

5.4.2.1 The basic model again 402

5.4.2.2 A special case – Ca2+ as necessary cofactor for closing 403

5.4.2.3 Expanding the basic model – Ca2+ affecting a voltage-independent step 404

5.5 Summing up 405

6. Quantifying the action: comparing the metal ions 407

6.1 Steady-state parameters are equally shifted 407

6.2 Different metal ions cause different shifts 408

6.3 Different metal ions slow gating differently 410

6.4 Block of ion channels 412

7. Locating the sites of action 412

7.1 Fixed surface charges involved in screening 413

7.2 Binding sites 413

7.2.1 Group 2 ions 414

7.2.2 Group 12 ions 414

8. Conclusions and perspectives 415

9. Appendix 416

10. Acknowledgements 418

11. References 418

Metal ions affect ion channels either by blocking the current or by modifying the gating. In the present review we analyse the effects on the gating of voltage-gated channels. We show that the effects can be understood in terms of three main mechanisms. Mechanism A assumes screening of fixed surface charges. Mechanism B assumes binding to fixed charges and an associated electrostatic modification of the voltage sensor. Mechanism C assumes binding and an associated non-electrostatic modification of the gating. To quantify the non-electrostatic effect we introduced a slowing factor, A. A fourth mechanism (D) is binding to the pore with a consequent pore block, and could be a special case of Mechanisms B or C. A further classification considers whether the metal ion affects a single site or multiple sites. Analysing the properties of these mechanisms and the vast number of studies of metal ion effects on different voltage-gated ion channels we conclude that group 2 ions mainly affect channels by classical screening (a version of Mechanism A). The transition metals and the Zn group ions mainly bind to the channel and electrostatically modify the gating (Mechanism B), causing larger shifts of the steady-state parameters than the group 2 ions, but also different shifts of activation and deactivation curves. The lanthanides mainly bind to the channel and both electrostatically and non-electrostatically modify the gating (Mechanisms B and C). With the exception of the ether-à-go-go-like channels, most channel types show remarkably similar ion-specific sensitivities.

1. Introduction 71

2. Electron transfer in PS II 72

3. (Mn)4cluster and mechanism of water oxidation 73

4. Organization and structure of the protein subunits 75

5. Organization of chlorophylls and redox active cofactors 81

6. Implications arising from the structural models 82

7. Perspectives 84

8. Acknowledgements 86

9. Addendum 86

10. References 87

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3·7/3·8 Å, have allowed assignments to be made to the redox active cofactors involved in the light-driven water–plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the D1 protein and approximately 7 Å from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.

1. Introduction 308

2. Plan of this article 312

3. Natural mechanisms of development of novel protein functions 313

3.1 Divergence 313

3.2 Recruitment 316

3.3 ‘Mixing and matching’ of domains, including duplication/oligomerization, and domain swapping or fusion 316

4. Classification schemes for protein functions 317

4.1 General schemes 317

4.2 The EC classification 318

4.3 Combined classification schemes 319

4.4 The Gene Ontology Consortium 321

5. Methods for assigning protein function 321

5.1 Detection of protein homology from sequence, and its application to function assignment 321

5.2 Detection of structural similarity, protein structure classifications, and structure/function correlations 326

5.3 Function prediction from amino-acid sequence 327

5.3.1 Databases of single motifs 328

5.3.2 Databases of profiles 329

5.3.3 Databases of multiple motifs 330

5.3.4 Precompiled families 331

5.3.5 Function identification from sequence by feature extraction 331

5.4 Methods making use of structural data 332

6. Applications of full-organism information: inferences from genomic context and protein interaction patterns 334

7. Conclusions 335

8. Acknowledgements 335

9. References 335

The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein–protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.

1. Chaperonin action – an overview 230

2. Polypeptide binding – an essential action 235

3. Recognition of non-native polypeptide – role of hydrophobicity 236

4. Crystallographic analyses of peptide binding 237

5. Topology and secondary and tertiary structure of bound substrate polypeptide – fluorescence, hydrogen exchange and NMR studies 239

6. Binding by GroEL associated with a putative unfolding action 242

7. A potential action of substrate unfolding driven by ATP/GroES binding 245

8. Folding in theciscavity 247

9. GroEL–GroES-mediated folding of larger substrate proteins by atransmechanism 249

10. Prospects for resolving the conformations and fate of polypeptide in the chaperonin reaction 251

11. References 252

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL–GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.

1. Introduction 430

2. Transformations of the RecA filament 431

2.1 The different forms of the RecA filament 431

2.2 Orientation and position of the RecA monomers in the active filament 433

2.3 Transmission of structural information along the filament 433

3. RecA-induced DNA deformations 435

3.1 Characteristics of RecA-bound DNA 435

3.2 Stretching properties of double-stranded DNA 436

3.3 DNA bound to architectural proteins 437

3.4 Implications for RecA-induced DNA deformations 438

3.5 Axial distribution of the DNA stretching deformation 438

4. Contacts between RecA and the DNA strands 440

4.1 The DNA-binding sites 440

4.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 442

5. Strand arrangement during pairing reorganization 444

5.1 Hypotheses for DNA strand association 444

5.2 Association via major or minor grooves 446

5.3 Post-strand exchange geometries 446

6. Conclusion 447

7. Acknowledgments 448

8. References 448

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

1. History 342

2. Activation barriers 343

2.1 Redox potentials 344

2.2 Reorganization energy 344

3. Electronic coupling 345

4. Ru-modified proteins 348

4.1 Reorganization energy 349

4.1.1 Cyt c 349

4.1.2 Azurin 350

4.2 Tunneling timetables 352

5. Multistep tunneling 357

6. Protein–protein reactions 359

6.1 Hemoglobin (Hb) hybrids 359

6.2 Cyt c/cyt b5 complexes 360

6.3 Cyt c/cyt c peroxidase complexes 360

6.4 Zn–cyt c/Fe–cyt c crystals 361

7. Photosynthesis and respiration 362

7.1 Photosynthetic reaction centers (PRCs) 362

7.2 Cyt c oxidase (CcO) 364

8. Concluding remarks 365

9. Acknowledgments 366

10. References 366

Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these ‘currents of life’. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein–protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions; it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.

1. Introduction 92

2. Methods and models 93

2.1 Density Functional Theory 93

2.2 Chemical models 98

3. Examples of mechanisms studied 104

3.1 Photosystem II 105

3.2 Cytochrome c oxidase 108

3.3 Manganese catalase 112

3.4 Ribonucleotide reductase 114

3.5 Methane mono-oxygenase 119

3.6 Methyl coenzyme M reductase 122

3.7 Intra- and extradiol dioxygenases 124

3.8 Tyrosinase and catechol oxidase 126

3.9 Amino-acid hydroxylases 130

3.10 Isopenicillin N synthase 132

3.11 Cytochrome c peroxidase 134

3.12 Copper-dependent amine oxidase 136

3.13 Galactose oxidase 138

4. Summary and conclusions 138

5. Acknowledgements 140

6. References 140

The study of metalloenzymes using quantum chemical methods of high accuracy is a relatively new field. During the past five years a quite good understanding has been reached concerning the methods and models to be used for these systems. For systems containing transition metals hybrid density functional methods have proven both accurate and computationally efficient. A background on these methods and the accuracy achieved in benchmark tests are given first in this review. The rest of the review describes examples of studies on different metalloenzymes. Most of these examples describe mechanisms where dioxygen is either formed, as in photosystem II, or cleaved as in many other enzymes life cytochrome c oxidase, ribonucleotide reductase, methane mono-oxygenase and tyrosinase. In the descriptions below high emphasis is put on the actual determination of the transition states, which are the key points determining the mechanisms.