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Cytochrome b561 (cyt b561) proteins are members of the recently identified eukaryotic ascorbate reducible protein family named CYBASC (CYtochrome B, ASCorbate reducible). CYBASC proteins are di-heme-b-containing membrane proteins that catalyze the transmembrane electron transfer from ascorbate. The function of the CYBASC proteins has been correlated with ascorbate recycling and/or iron facilitation uptake. Therefore, investigations on this family are of great interest as ascorbate is one of the most powerful antioxidants and iron is essential for cell survival both in animals and plants. As the amino acid sequence conservation of animal and plant CYBASC proteins is relatively high, all CYBASC members are proposed to share the same structural motifs. However, no three-dimensional structure of any representative member of the CYBASC family has been determined to date. In the Arabidopsis thaliana (A. thaliana) genome, two complete putative CYBASC open reading frames (ORFs), artb561-a and artb561-b were identified. In this thesis, these two A. thaliana CYBASC ORFs, encoding for Acytb561-A and Acytb561-B proteins respectively, were investigated and obtained main results are listed. 1. A. thaliana CYBASC proteins were heterologously produced in Pichia pastoris and Escherichia coli and purified by a single-step immobilized metal affinity chromatography (IMAC). To facilitate detection and purification, the recombinant A. thaliana CYBASC proteins were produced in both expression systems with the histidine affinity tag. Pure and stable preparations of the cytochromes were obtained via a single-step IMAC in sufficient amounts to perform biochemical characterizations. 2. Detergent solubilized recombinant Acytb561-A and Acytb561-B are dimers. As previously suggested for other CYBASC proteins, analytical gel filtration experiment suggested that both detergent solubilized cytochromes are dimers. 3. Spectroscopic features of Acytb561-B differed from those of previously described bovine chromaffin granule cyt b561. A distinctive feature of the first identified CYBASC protein, the cyt b561 from bovine chromaffin vesicles of adrenal medulla (Bcytb561-CG), is that its differential visible absorbance spectra (visible-spectra) revealed an asymmetric α-band with a maximum at 562 nm and a clear shoulder at 557 nm. This feature was recently used to discriminate CYBASC proteins from not-CYBASC proteins. However, in this thesis, it is shown for the first time that not all CYBASC proteins display in their reduced-minus-oxidized visible-spectra an asymmetric α- band and therefore, this feature can not be used as a discriminating CYBASC characteristic. 4. Ascorbate dependent reduction of the A. thaliana CYBASC proteins is inhibited by diethylpyrocarbonate (DEPC). As previously reported for the Bcytb561-CG, the ascorbatedependent reduction of the A. thaliana CYBASC proteins was inhibited by DEPC treatment. In addition, the ‘ascorbate protectant’ effect against DEPC that was observed on the Bcytb561-CG was also observed on the Acytb561-A and Acytb561-B proteins. Furthermore, as the physiological electron donor of all CYBASC proteins is supposed to be ascorbate, ascorbate-affinity of Acytb561- A and Acytb561-B was monitored and was found to be in the same range of the one of the Bcytb561- CG. 5. A. thaliana CYBASC proteins are Fe3+-chelate reductases. Recently, the Fe3+-chelate reductase activity of various CYBASC proteins was presented. In this thesis, it is shown that also both A. thaliana CYBASC proteins reduced Fe3+-chelates such as Fe3+-EDTA and Fe3+-citrate. Consistently, heme potentiometric reductive-oxidative titration of purified Acytb561-A and Acytb561-B indicated that the midpoint potential of the two heme centres of both cytochromes was lower than the one of those Fe3+-chelates. The values of both heme centre potentials of Acytb561-A and Acytb561-B are also consistent with the observation that both cytochromes were only partially reducible by ascorbate and were fully reduced with the non-physiological reductant Na-dithionite. In summary, this work describes the heterologous production, purification and initial characterizations of two distinct CYBASC proteins from A. thaliana: Acytb561-A and Acytb561-B. Biochemical characterization of these cytochromes showed that the shape of the α-band in the differential spectra is not a discriminating factor for CYBASC proteins but it is likely the DEPC sensitivity and the Fe3+-chelate reductase activity. Establishment of a purification strategy to obtain sufficient amounts of monodispersed and stable A. thaliana CYBASC proteins has also enabled initial screening of three dimensional crystallization conditions which are a prerequisite for a deeper understanding of this new eukaryotic redox enzyme family.
High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and punfication of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptideslproteins by sizeexclusion chromatography and monitoring the hydrophilicitykydrophobicity of amphipathic cr-helical peptides, a vital precursor Tor the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins. Key Words: Peptides; proteins; size-exclusion chromatography (SEC); anion-exchange chromatography (AEX); cation-exchange chromatography (CEX); mixed-mode hydrophilic interaction chromatography (HIL1C)/cation-exchange chromatography (CEX); reversed-phase high-performance liquid chromatography (RP-HPLC); preparative RP-HPLC of peptides and proteins; amino acid side-chain hydrophilicitylhydrophobicity coefficients; amino acid U-helical propensity values; amino acid side-chain stability coefficients
Lepidoptera phylogeny and systematics : the state of inventorying moth and butterfly diversity
(2007)
The currently recognized robust support for the monophyly of the Lepidoptera (and the superorder Amphiesmenoptera comprising Lepidoptera + Trichoptera) is outlined, and the phylogeny of the principal lineages within the order is reviewed succinctly. The state of the taxonomic inventory of Lepidoptera is discussed separately for ‘micro-moths’, ‘macro-moths’ and butterflies, three assemblages on which work has followed historically somewhat different paths. While currently there are about 160,000 described species of Lepidoptera, the total number of extant species is estimated to be around half a million. On average, just over one thousand new species of Lepidoptera have been described annually in recent years. Allowing for the new synonyms simultaneously established, the net increase in species numbers still exceeds 800/year. Most of the additions are foreseeable in the micro-moth grade, but even for butterflies ca 100 species are added annually. Examples of particularly interesting new high-rank taxa that have been described (or whose significance has become realized) since the middle of the 20th century include the non-glossatan lineages represented by Agathiphaga and Heterobathmia and the heteroneuran families Andesianidae, Palaephatidae, Hedylidae and Micronoctuidae. Some thoughts on how present and future systematic lepidopterology might be prioritised are presented.
The taxonomy, diversity, and distribution of the aquatic insect order Trichoptera, caddisflies, are reviewed. The order is among the most important and diverse of all aquatic taxa. Larvae are vital participants in aquatic food webs and their presence and relative abundance are used in the biological assessment and monitoring of water quality. The species described by Linnaeus are listed. The morphology of all life history stages (adults, larvae, and pupae) is diagnosed and major features of the anatomy are illustrated. Major components of life history and biology are summarized. A discussion of phylogenetic studies within the order is presented, including higher classification of the suborders and superfamilies, based on recent literature. Synopses of each of 45 families are presented, including the taxonomic history of the family, a list of all known genera in each family, their general distribution and relative species diversity, and a short overview of family-level biological features. The order contains 600 genera, and approximately 13,000 species.
Presented herein is the first morphological analysis of turtle relationships to examine the monophyly of many turtle groups by using only single species as terminals and by integrating a large number of primitive fossil taxa. The data matrix consists of 136 osteological parsimony informative characters with 169 derived character states for 45 fossil and 22 living species of the clade TESTUDINATA. The results corroborate the monophyly of a large number of previously hypothesized clades, but refute the accepted hypothesis regarding the basal split of living turtles. In particular, the primitive turdes Proterochersis robusta, Kayentachelys aprix, Mongolochelys efremovi, Meiolania platyceps, and Kallokibotion bajazidi are removed from their current position as crown turtles and placed along the phylogenetic stem of this clade. The age of the turtle crown is thereby adjusted from the Late Triassic to the Late Jurassic, which is relevant to testing molecular clock hypotheses. This revised topology has important implications for the evolution of several character complexes, because it implies that the common ancestor of all living turtles must have had a partially braced brain case and a primitive trochlear mechanism. Other noteworthy conclusions include the tentative exclusion of protostegids from CHELONIOIDEA, the placement of Platysternon megacephalum outside of CHELYDRIDAE, and the tentative interpretation of Sandownia harrisi as a basal eucryptodire.
The impact of naval sonar on beaked whales is of increasing concern. In recent years the presence of gas and fat embolism consistent with decompression sickness (DCS) has been reported through postmortem analyses on beaked whales that stranded in connection with naval sonar exercises. In the present study, we use basic principles of diving physiology to model nitrogen tension and bubble growth in several tissue compartments during normal div ng behavior and for several hypothetical dive profiles to assess the risk of DCS. Assuming that normal diving does not cause nitrogen tensions in excess of those shown to be safe for odontocetes, the modeling indicates that repetitive shallow dives, perhaps as a consequence of an extended avoidance reaction to sonar sound, can indeed pose a risk for DCS and that this risk should increase with the duration of the response. If the model is correct, then limiting the duration of sonar exposure to minimize the duration of any avoidance reaction therefore has the potential to reduce the risk of DCS.
The former and current distribution of the quokka, Setortix brachyurus, was mapped from published and all available unpublished records. At the time of European settlement the quokka was widespread and abundant and its distribution encompassed an area of approximatelyThe former and current distribution of the quokka, Setortix brachyurus, was mapped from published and all available unpublished records. At the time of European settlement the quokka was widespread and abundant and its distribution encompassed an area of approximately 41 200 km2 of south-west Western Australia inclusive of two offshore islands, Bald Island and Rottnest Island. Historical reports indicated an extensive population decline occurred in the 1930s. The decline continued, with a previously undocumented decline apparent in the period from 1980 to 1992. However, this decline may be an artefact of the time scales used for mapping and may well equate with a previously reported decline lor a suite of south -west mammals in the 1970s. By 1992 the quokka´s distribution had been reduced to an area of approximately 17800 km2. An increased awareness of the presence of the quokka on the mainland has resulted in numerous reportings of quokka presence since 1992, has confimled the existence of several populations at the northern extent of the quokka´´s known geographic range and indicated the cmrent, 2005, distribution to be similar to that in 1992. However, survey and population estimates at six of these mainland locations from the northem jarrah forest indicated low abundance. There have been no population estimates elsewhere on the mainland. Two populations have been reported tiom the Swan Coastal Plain, but neither has been confirmed extant. Predation by the introduced fox, Vulpes vulpes, is implicated as a major cause of the quokka´s initial decline, while ongoing predation, habitat destruction and modification through altered tire regimes have contributed to the continued decline. Specific conservation management actions are recommended, namely: (i) Implementing an active adaptive management program in the northern jarrah forest to determine quokka population response to habitat manipulation through the use of fIre, fox baiting and pig control; (ii) Surveying the Stirling fumge and Green Range populations with emphasis placed on determining population size and population genetic structure; (iii) Surveying the reported occurrences from the Swan Coastal Plain, with emphasis on unambiguously determining presence. If confirmed, priority should he directed to assessing population size and determining the management requirements to ensure persistence of the population; (iv) Surveying southem forest and south coast populations to assess quokka population size, the extent of movement between sllbpopulations and assessment of the range of habitat types used by quokkas. The latter should be combined with spatial analyses of known extant populations and suitable and potentially suitable habitat; (v) Determining the role of tire in establishing and maintaining preferred habitat of southern forest and south coast populations; and (vi) Establishing a program to assess the potential effects from management operations.
Techniques for collecting, handling, preparing, storing and examining small molluscan specimens
(2007)
Micromolluscs are small-sized molluscs (< 5 mm), and include the great majority of undescribed molluscan taxa. Such species require special collecting, sorting and handling techniques and different storage requirements to those routinely used for larger specimens. Similarly, the preparation of shells, opercula, radulae and animals poses some challenges for scanning electron microscopy (SEM). An overview of experiences with various techniques is presented, both positive and negative. Issues discussed include those relating to storage of dry specimens and interaction of specimens with glass, gelatine and paper products, handling techniques and storage in various fluids. Techniques for cleaning shells for SEM are described and compared, as well as those for radular extraction. The interactions of chemicals used for the dissolution of tissue with calcareous micromolluscs are described. Methods for handling and mounting small radulae for SEM are detailed and brief guides to SEM and light photography are given. An appendix listing details of frequently-used chemicals is provided.
Shaped by some of the most dramatic tectonic events of the Cenozoic, the parts of southern and eastern Asia that have become known as the Oriental faunal region comprise vast areas of great geological complexity and ecological diversity. One of the four major groups of terrestrial elapid snakes in this region is the genus Bungarus. These nocturnal and predominantly ophiophagous snakes are widely known as kraits and are an important cause of snakebite mortality throughout their wide range that extends from Afghanistan to Vietnam and eastern China, and south to the Indonesian islands of Java and Bali. Although present on Borneo, kraits have not been found on any island of the Philippines, nor on Lesser Sunda Islands east of Bali. Despite their medical significance and the great importance of Bungarus toxins as tools in neuropharmacology, krait systematics and taxonomy have remained largely unstudied. Twelve species of Bungarus were recognized at the beginning of the present study. Many of these are rare in collections, and most aspects of their biology are unknown. While some species are highly distinct, most kraits are conservative morphologically, rendering molecular methods invaluable for the study of their diversity and biogeography. This study is the first to address the relationships within Bungarus and the historical biogeography of kraits based on molecular evidence. I inferred phylogeographic relationships based on analyses of new nucleotide sequences of the entire mitochondrial cytochrome b gene of 51 kraits and partial NADH dehydrogenase subunit 4 sequences of 40 kraits which I analyzed together with a representative sample of 32 published elapid and non-elapid outgroup taxa using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods. I then used the recovered phylogeny to investigate the evolution of selected morphological characters and, together with collections-based geographical distribution information, in dispersal-vicariance analyses with models of variable taxonomic and biogeographic complexity. The phylogenetic analyses demonstrate that the current taxonomy of kraits does not adequately represent either the relationships or the genetic diversity in this genus. In contrast, I identified monophyletic groups that are congruent with recognized biogeographic units as well as extensive ecomorph evolution and morphologically cryptic speciation. The following additional conclusions are collectively supported by the mitochondrial phylogeny and morphological as well as biochemical synapomorphies: (1) Kraits are monophyletic with respect to the remaining taxa of the Elapidae; (2) Bungarus flaviceps and Bungarus bungaroides form the monophyletic sister clade of a clade formed by B. fasciatus, black-and-white-banded, and uniformly black taxa; (3) the remaining taxa are divisible into two sister clades, the South Asian species (Bungarus sindanus (Bungarus caeruleus, Bungarus ceylonicus)) vs. Himalayan, Burmese, Southeast and East Asian taxa; (4) within the latter, Burmese taxa form the sister clade to Southeast and East Asian taxa; (5) the widespread and medically significant species Bungarus candidus and Bungarus multicinctus are paraphyletic. The results of this study highlight the importance of vicariant geological events and sea level fluctuations for the cladogenesis of kraits. Events of particular importance in the evolution of kraits include the uplift of the Indo-Burman ranges (Arakan-Naga Hills) which separated black-and-white banded kraits in India and Southeast Asia, and the uplift of mountain ranges in Yunnan, China (e.g., the Gaoligong Shan), which coincided with lineage separation in two distantly related clades of kraits. Alternating dispersal and vicariance events due to Pleistocene climatic and sea level changes have caused complex phylogeographic patterns in kraits in Southeast Asia. Zones of contact between closely related evolutionary lineages of the B. candidus complex are identified in Thailand, Vietnam, and southern China (Hainan). Within this complex, two main clades are revealed. One includes populations from the Southeast Asian mainland and is in contact with B. multicinctus in southern China. The other consists of populations from Thailand, southern Vietnam, Java, and Bali. The phylogeny as well as genetic distances suggest a scenario in which a Pleistocene southward dispersal of B. candidus to Sumatra, Java, and Bali during times of low sea levels was temporarily interrupted by vicariant events (rising sea levels, especially flooding of the Malacca Strait between Sumatra and the Malay Peninsula, and of the Bali Strait between Java and Bali). In this context, the close phylogenetic relationship between haplotypes from southern Vietnam and those from Java and Bali suggests that "southern" B. candidus dispersed directly via colonization of the widely receded South Chinese Sea, and not by taking a detour via the Malay Peninsula and Thailand, which were already inhabited by other populations of B. candidus. Using these phylogenetic estimates as the framework for a study on the diversity and evolution of krait venom components, I applied biochemical and molecular genetic approaches to identify and quantify polypeptide and protein toxins in krait venom, focusing on the distribution and molecular evolution of alpha-bungarotoxin, an irreversible competitive antagonist of nicotinic acetylcholine receptors with an exceptionally high applied significance as a receptor probe. I was specifically interested in the medically relevant question of intraspecific and interspecific variability in toxin diversity, and whether receptor-binding postsynaptic toxins evolve at rates different from those of presynaptic neurotoxins like beta-bungarotoxin, which act by destroying the nerve terminal and are believed to exhibit hypervariable functional diversification due to an accelerated mode of molecular evolution. In the context of this question, I isolated and purified the major lethal neurotoxins from B. candidus venoms by sequential steps of liquid chromatography for structural and functional characterization studies. Cloning and sequence analysis of toxin-coding genomic DNAs showed that the gene encoding the alpha-bungarotoxin alanine-31 variant, originally isolated from B. multicinctus venom, is widely present and highly conserved in multiple populations of B. candidus and is expressed as the principal postsynaptic neurotoxin at least in Javan B. candidus. In addition to the widespread presence of genomic DNAs encoding the alpha-bungarotoxin alanine-31 variant, the present study also revealed the partial genes of three novel alpha-bungarotoxin isoforms in addition to the previously known alanine-31 and valine-31 variants, all of which share an invariant exon 3 coding region. While alpha-bungarotoxin is the principal postsynaptic neurotoxin of Taiwanese B. multicinctus and Javan B. candidus, the main postsynaptic neurotoxin of Thai B. candidus both by quantity and lethality was a novel polypeptide of similar toxicity with a mass of 8030 Da and 73 amino acid residues, whose characterization at the genetic and protein levels revealed a novel subgroup of krait neurotoxins, here named alpha-delta-bungarotoxins and represented by four sequences from Bungarus caeruleus and B. candidus. alpha-delta-Bungarotoxins share high sequence homology with alpha-bungarotoxins but the purified, 8030 Da alpha-delta-bungarotoxin-1 exhibits only reversible, low affinity binding to nicotinic receptors and high site-selectivity for the acetylcholine binding site at the alpha-delta-subunit interface of the receptor. These properties render alpha-delta-bungarotoxin not only the first snake long-chain neurotoxin with reversible binding and binding-site selectivity, but also an exciting natural tool with which to address structure-function relationships at the subunit interfaces of the human receptor. The results of comparisons of the number of non-synonymous nucleotide substitutions per nonsynonymous site (dN) to the number of synonymous nucleotide substitutions per synonymous site (dS) strongly suggest that positive selection is acting on exon 2 of the alpha-bungarotoxin and probably also of the alpha-delta-bungarotoxin genes. In addition, the numbers of nucleotide substitutions per site of intron (dI) compared to the dS value of the toxin-coding exon regions provide strong evidence for accelerated molecular evolution in exon 2 of alpha-delta-bungarotoxins —whose value of dI is only one-eighth of the value of dS—whereas the hypothesis of accelerated evolution is rejected for 13 unique genomic DNAs encoding five alpha-bungarotoxin isoforms from B. candidus and B. multicinctus....
Glyptostrobus Endlicher is well represented in early Early Cretaceous to Pleistocene deposits in the middle to high latitudes of North America and Eurasia. Although the taxonomy and nomenclature of the genus is complicated, the fossil record indicates Glyptostrobus was represented by a small number of species. The genus first appears in Aptian age deposits from western Canada and Greenland, and achieved a wide distribution early in its evolutionary history. Exchange of Glyptostrobus between Asia and North America occurred across the Spitsbergen and Beringian corridors, which were functional about 110 and 100 million years ago, respectively The Late Cretaceous fossil record of Glyptostrobus shows that the genus had spread into Russia, China and the shores of the Turgai Strait. By the early Tertiary, Glyptostrobus was a prominent constituent of the polar broad-leaved deciduous forests. Paleocene age deposits across western Canada and the United States indicate the genus was present in great abundance in the lowland warm temperate and subtropical forests east of the Rocky Mountains. The broad distribution in North America and Russia during the Paleocene and Eocene indicates that Glyptostrobus grew and reproduced under a diverse range of climatic and environmental conditions, including the cold and unique lighting conditions of the polar latitudes. The presence of Glyptostrobus in Europe indicates the North Atlantic land bridges that extended between North America and Eurasia (Fennoscandia) and Europe during the early Tertiary were used. In Europe, extensive Glyptostrobus dominated swan1ps occupied the Central European Depression during the late Tertiary. Increasing global aridity and cooling, as well as landscape stabilization together with increasing competition for resources and habitat by representatives of the Pinaceae, seem to have forced the genus out of North America, Europe and most of Asia during the Miocene and Pliocene. In Japan, Glyptostrobus persisted until the early Pleistocene. After the early Pleistocene extinction in Japan, Glyptostrobus reappeared in southeastern China. Details of the taxonomic and biogeographic history of Glyptostrobus are examined.
The chemiosmotic theory suggested by Peter Mitchell (Mitchell, 1961, Nature 191:144-148; see Mitchell, 1979, Science 206:1148-1159 for review) postulated that the energy released upon the oxidation of electron donor substrates is transiently stored as electrochemical proton potential, delta-p across energy-transducing membranes, which acts then as the driving force for the ATP synthesis. Membrane protein complexes can both generate and utilise a transmembrane electrochemical proton potential, either by transmembrane proton transfer or by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane. The dihaem-containing membrane protein complex quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes apparently combines both of these mechanisms (Haas et al, 2005, Biochemistry 44:13949-13961; Lancaster et al, 2005, PNAS 102:18860–18865; Mileni et al, 2005, Biochemistry 44:16718-16728; Madej et al, 2006, EMBO J 25:4963-4970). QFR is the terminal enzyme of anaerobic fumarate respiration that allows bacteria to use fumarate as the terminal electron acceptor (Kröger, 1978, Biochim Biophys Acta 505:129-45; Lancaster, 2004, In: Respiration in Archaea and Bacteria Volume 1:57-85). QFR couples the two-electron reduction of fumarate to succinate to the two-electron oxidation of quinol to quinone. QFR contains two haem b groups bound by the transmembrane subunit C, which are termed the ‘proximal haem’, bP, and the ‘distal haem’, bD, according to the relative proximity to the hydrophilic subunits A and B (Lancaster et al, 1999, Nature 402:377-85). The two-electron transfer via the two haem groups has been proposed (Lancaster, 2002, Biochimica et Biophysica Acta 1565:215-231) and demonstrated (Madej et al, 2006, EMBO J 25:4963-4970) to be coupled to a compensatory, parallel transfer of two protons via a transmembrane proton transfer pathway. The two most prominent constituents of the proposed pathway were suggested to be the haem bD ring C propionate and the side chain of amino-acid residue Glu C180, after which the proton transfer pathway was named the ‘E-pathway’ (Lancaster, 2002, Biochimica et Biophysica Acta 565:215-231). The essential role of Glu C180 was supported by site-directed mutagenesis and structural and functional characterization of the enzyme E180Q, where the Glu C180 was replaced with a Gln residue (Lancaster et al, 2005, PNAS 102:18860–18865). Moreover, multiconformer continuum electrostatics (MCCE) calculations (Haas and Lancaster 2004, Biophys J 87:4298-4315) and Fouriertransformed infrared (FTIR) spectroscopy experiments (Haas et al, 2005, Biochemistry 44:13949-13961) indicated the Glu C180 side chain to undergo a combination of a conformational change and protonation upon haem reduction. The contribution of haem bD propionate is less clear, however, a combination of 13C labelling of the haem propionates with redox-induced FTIR experiments (Mileni et al, 2005, Biochemistry 44:16718-16728) and MCCE calculations (Haas and Lancaster, 2004, Biophys J 87:4298-4315) support a change in protonation, possibly accompanied by a change in environment upon haem reduction. These experiments and their results strongly support the existence of the ‘E-pathway’ which is transiently open during the reduction of the haem groups and blocked in the oxidized state of the enzyme (Lancaster, 2002b, Biochim Biophys Acta 1565:215-231). All available crystal structures of the QFR, however, are those of the oxidized enzyme. Therefore, it is advantageous to perform simulations of various redox states of the enzyme to determine for instance, how the side-chain of Glu C180 and haem bD ring C propionate behave upon changes of the redox states of the haem groups and why is the ‘E-pathway’ blocked in the oxidized state of the enzyme. Although the distal haem ring C propionate and Glu C180 were identified as the most prominent components of the proton transfer pathway, it was not clear, on the basis of the structure, how proton transfer could occur between them. In addition, two constituents are not enough to span the membrane region and the additional participants in the proton transfer pathway must be identified. Since an atomistic investigation of proton transfer in this system is not yet possible experimentally, I used available theoretical methods such as classical molecular dynamics (MD) simulation (Alder and Wainwright, 1959, J Phys Chem 31:459-466; McCammon et al, 1977, Nature 267:585-590) and Q-HOP molecular dynamics (Q-HOP MD) simulation (Lill and Helms, 2001, J Chem Phys 115:7993-8005) to investigate the postulated mechanism of electron coupled proton transfer in QFR. MD simulations allowed us to move away from static difference pictures obtained from FTIR experiments and MCCE calculations. The advantage of the MD simulations over the experiments and the simulations performed so far is that the time-dependent properties could now be analyzed. The behaviour of various residues and their side-chains and any environmental changes may be directly observed during MD simulations. Although classical MD simulations cannot be used to study proton transfer reactions, they can provide information on formation of configurations that would allow either direct proton transfer between donor and acceptor residues or indirect proton transfer mediated by water molecules. To avoid the static protonation of residues which is inherent in classical MD simulations, Q-HOP MD simulations were performed which explicitly describe proton transfer reactions by allowing the change of the protonation state of residues ‘on the fly’. The structures obtained after classical molecular dynamics simulations ....