Structural evidence for intermediates during O2 formation in photosystem II.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation.

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens ('substrate waters') in the various intermediates (Si states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn4CaO5) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H218O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the Si state and are hence referred to as the fast (Wf) and the slow (WS) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S2 state, under special experimental conditions, two different rates of WS exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn4CaO5 cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as WS in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.

Structures of the intermediates of Kok's photosynthetic water oxidation clock.

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Alternative Mechanism for O2 Formation in Natural Photosynthesis via Nucleophilic Oxo-Oxo Coupling.

O2 formation in photosystem II (PSII) is a vital event on Earth, but the exact mechanism remains unclear. The presently prevailing theoretical model is "radical coupling" (RC) involving a Mn(IV)-oxyl unit in an "open-cubane" Mn4CaO6 cluster, which is supported experimentally by the S3 state of cyanobacterial PSII featuring an additional Mn-bound oxygenic ligand. However, it was recently proposed that the major structural form of the S3 state of higher plants lacks this extra ligand, and that the resulting S4 state would feature instead a penta-coordinate dangler Mn(V)=oxo, covalently linked to a "closed-cubane" Mn3CaO4 cluster. For this proposal, we explore here a large number of possible pathways of O-O bond formation and demonstrate that the "nucleophilic oxo-oxo coupling" (NOOC) between Mn(V)=oxo and µ3-oxo is the only eligible mechanism in such a system. The reaction is facilitated by a specific conformation of the cluster and concomitant water binding, which is delayed compared to the RC mechanism. An energetically feasible process is described starting from the valid S4 state through the sequential formation of peroxide and superoxide, followed by O2 release and a second water insertion. The newly found mechanism is consistent with available experimental thermodynamic and kinetic data and thus a viable alternative pathway for O2 formation in natural photosynthesis, in particular for higher plants.

Solar energy conversion by photosystem II: principles and structures.

Photosynthetic water oxidation by Photosystem II (PSII) is a fascinating process because it sustains life on Earth and serves as a blue print for scalable synthetic catalysts required for renewable energy applications. The biophysical, computational, and structural description of this process, which started more than 50 years ago, has made tremendous progress over the past two decades, with its high-resolution crystal structures being available not only of the dark-stable state of PSII, but of all the semi-stable reaction intermediates and even some transient states. Here, we summarize the current knowledge on PSII with emphasis on the basic principles that govern the conversion of light energy to chemical energy in PSII, as well as on the illustration of the molecular structures that enable these reactions. The important remaining questions regarding the mechanism of biological water oxidation are highlighted, and one possible pathway for this fundamental reaction is described at a molecular level.

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function.

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 → S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae.

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H2 yield.

Assessment of the manganese cluster's oxidation state via photoactivation of photosystem II microcrystals.

Knowledge of the manganese oxidation states of the oxygen-evolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)3Mn(IV) or Mn(III)Mn(IV)3 for the S2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster's oxidation states.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Reversible Structural Isomerization of Nature's Water Oxidation Catalyst Prior to O-O Bond Formation.

Photosynthetic water oxidation is catalyzed by a manganese-calcium oxide cluster, which experiences five "S-states" during a light-driven reaction cycle. The unique "distorted chair"-like geometry of the Mn4CaO5(6) cluster shows structural flexibility that has been frequently proposed to involve "open" and "closed"-cubane forms from the S1 to S3 states. The isomers are interconvertible in the S1 and S2 states, while in the S3 state, the open-cubane structure is observed to dominate inThermosynechococcus elongatus (cyanobacteria) samples. In this work, using density functional theory calculations, we go beyond the S3+Yz state to the S3nYz• → S4+Yz step, and report for the first time that the reversible isomerism, which is suppressed in the S3+Yz state, is fully recovered in the ensuing S3nYz• state due to the proton release from a manganese-bound water ligand. The altered coordination strength of the manganese-ligand facilitates formation of the closed-cubane form, in a dynamic equilibrium with the open-cubane form. This tautomerism immediately preceding dioxygen formation may constitute the rate limiting step for O2 formation, and exert a significant influence on the water oxidation mechanism in photosystem II.

Water Oxidation by Pentapyridyl Base Metal Complexes? A Case Study.

The design of molecular water oxidation catalysts (WOCs) requires a rational approach that considers the intermediate steps of the catalytic cycle, including water binding, deprotonation, storage of oxidizing equivalents, O-O bond formation, and O2 release. We investigated several of these properties for a series of base metal complexes (M = Mn, Fe, Co, Ni) bearing two variants of a pentapyridyl ligand framework, of which some were reported previously to be active WOCs. We found that only [Fe(Py5OMe)Cl]+ (Py5OMe = pyridine-2,6-diylbis[di-(pyridin-2-yl)methoxymethane]) showed an appreciable catalytic activity with a turnover number (TON) = 130 in light-driven experiments using the [Ru(bpy)3]2+/S2O82- system at pH 8.0, but that activity is demonstrated to arise from the rapid degradation in the buffered solution leading to the formation of catalytically active amorphous iron oxide/hydroxide (FeOOH), which subsequently lost the catalytic activity by forming more extensive and structured FeOOH species. The detailed analysis of the redox and water-binding properties employing electrochemistry, X-ray absorption spectroscopy (XAS), UV-vis spectroscopy, and density-functional theory (DFT) showed that all complexes were able to undergo the MIII/MII oxidation, but none was able to yield a detectable amount of a MIV state in our potential window (up to +2 V vs SHE). This inability was traced to (i) the preference for binding Cl- or acetonitrile instead of water-derived species in the apical position, which excludes redox leveling via proton coupled electron transfer, and (ii) the lack of sigma donor ligands that would stabilize oxidation states beyond MIII. On that basis, design features for next-generation molecular WOCs are suggested.

Five-coordinate MnIV intermediate in the activation of nature's water splitting cofactor.

Nature's water splitting cofactor passes through a series of catalytic intermediates (S0-S4) before O-O bond formation and O2 release. In the second last transition (S2 to S3) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all MnIV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S4 How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S3') in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (>50%) in (i) chemical-modified cofactors in which Ca2+ is exchanged with Sr2+; the Mn4O5Sr cofactor remains active, but the S2-S3 and S3-S0 transitions are slower than for the Mn4O5Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S3' electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S3 signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S2 state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S3' cofactor is similar to the high spin form of the preceding S2 state, which contains a cuboidal Mn3O4Ca unit tethered to an external, 5-coordinate Mn ion (Mn4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn4. The interaction of ammonia with the cofactor is also discussed.

Bicarbonate-Mediated CO2 Formation on Both Sides of Photosystem II.

The effect of bicarbonate (HCO3-) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3- enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the QA quinone leads to the release of HCO3- from its binding site on the non-heme iron and (ii) the Em potential of the QA/QA•- couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the Em of QA decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with 13C isotope labeling of HCO3- allows donor- and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

Substrate water exchange in the S2 state of photosystem II is dependent on the conformation of the Mn4Ca cluster.

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the S2 state, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the S, S, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10 times faster in the S than in the S state, and that the S→ S conversion has at physiological temperature an activation barrier of 17 ± 1 kcal mol-1. Of the presently suggested S models, our findings are best in agreement with a water exchange pathway involving a S state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by a different equilibrium than in S2, and that the exchange of the fast substrate water in the S2 state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O-O bond formation in PSII are discussed.

More than protection: the function of TiO2 interlayers in hematite functionalized Si photoanodes.

Worldwide significant efforts are ongoing to develop devices that store solar energy as fuels. In one such approach, solar energy is absorbed by semiconductors and utilized directly by catalysts at their surfaces to split water into H2 and O2. To protect the semiconductors in these photo-electrochemical cells (PEC) from corrosion, frequently thin TiO2 interlayers are applied. Employing a well-performing photoanode comprised of 1-D n-Si microwires (MWs) covered with a mesoporous (mp) TiO2 interlayer fabricated by solution processing and functionalized with α-Fe2O3 nanorods, we studied here the function of this TiO2 interlayer by high-energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) spectroscopy, along with X-ray emission spectroscopy (XES) and standard characterization techniques. Our data reveal that the TiO2 interlayer not only protects the n-Si MW surface from corrosion, but that it also acts as a template for the hydrothermal growth of α-Fe2O3 nanorods and improves the photocatalytic efficiency. We show that the latter effect correlates with the presence of stable oxygen vacancies at the interface between mp-TiO2 and α-Fe2O3, which act as electron traps and thereby substantially reduce the charge recombination rate at the hematite surface.

XANES and EXAFS of dilute solutions of transition metals at XFELs.

This work has demonstrated that X-ray absorption spectroscopy (XAS), both Mn XANES and EXAFS, of solutions with millimolar concentrations of metal is possible using the femtosecond X-ray pulses from XFELs. Mn XAS data were collected using two different sample delivery methods, a Rayleigh jet and a drop-on-demand setup, with varying concentrations of Mn. Here, a new method for normalization of XAS spectra based on solvent scattering that is compatible with data collection from a highly variable pulsed source is described. The measured XANES and EXAFS spectra of such dilute solution samples are in good agreement with data collected at synchrotron sources using traditional scanning protocols. The procedures described here will enable XFEL-based XAS on dilute biological samples, especially metalloproteins, with low sample consumption. Details of the experimental setup and data analysis methods used in this XANES and EXAFS study are presented. This method will also benefit XAS performed at high-repetition-rate XFELs such as the European XFEL, LCLS-II and LCLS-II-HE.

Thomas John Wydrzynski (8 July 1947-16 March 2018).

With this Tribute, we remember and honor Thomas John (Tom) Wydrzynski. Tom was a highly innovative, independent and committed researcher, who had, early in his career, defined his life-long research goal. He was committed to understand how Photosystem II produces molecular oxygen from water, using the energy of sunlight, and to apply this knowledge towards making artificial systems. In this tribute, we summarize his research journey, which involved working on 'soft money' in several laboratories around the world for many years, as well as his research achievements. We also reflect upon his approach to life, science and student supervision, as we perceive it. Tom was not only a thoughtful scientist that inspired many to enter this field of research, but also a wonderful supervisor and friend, who is deeply missed (see footnote*).