International
Tables for
Crystallography
Volume I
X-ray absorption spectroscopy and related techniques
Edited by C. T. Chantler, F. Boscherini and B. Bunker

International Tables for Crystallography (2022). Vol. I. Early view chapter
https://doi.org/10.1107/S1574870720004772

Metalloproteins and systems of biological relevance

Richard W. Strangea*

aSchool of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom
Correspondence e-mail: rstrange@essex.ac.uk

This chapter provides an overview of the successful application of X-ray absorption spectroscopy (XAS) to biological systems. The advantages and limitations of XAS in this field are discussed, from the early synchrotron-radiation era to the recent advance of the X-ray free-electron laser. The synergy between XAS and crystallography is illustrated by several examples, focusing on metalloproteins of significant biological interest.

Keywords: metalloproteins; biological systems.

1. Introduction

This chapter is focused on the application of X-ray absorption spectroscopy (XAS) to study metal centres in biological systems and its synergy with protein crystallography. XAS (also XANES, EXAFS) has become a standard experimental technique since the availability in the early 1970s of tuneable-energy high-intensity X-rays radiated by electron synchrotron storage rings. The biological molecules that it has most often been used to study are the metalloproteins, a class of protein containing one or more metal centres that perform catalytic or structural functions. In 1975, Kincaid and coworkers and Schulman and coworkers demonstrated the potential of these X-ray sources for XAS of biological systems, with studies on copper and nickel tetraphenylporphyrin and methemoglobin and the iron–sulfur protein rubredoxin (Kincaid et al., 1975[link]; Shulman et al., 1975[link]). Over the next five years, biological applications of XAS (BioXAS) grew rapidly, embracing metalloproteins with functions as important and diverse as nitrogen fixation, gas binding and transport, respiration, redox chemistry, hydroxylation and halogenation, electron transfer, metal storage and others. These subjects included the MoFe protein of nitrogenase (Cramer, Hodgson et al., 1978[link]), O2 and CO coordination in hemoglobin (Eisenberger et al., 1978[link]), the oxidation states of copper in CuZn bovine superoxide dismutase (Blumberg et al., 1978[link]), the iron sites in oxidized and reduced cytochrome c (Labhardt & Yuen, 1979[link]), the type I copper proteins (Tullius et al., 1978[link]), iron coordination in P450 and chloroperoxidase (Cramer, Dawson et al., 1978[link]), the iron core in ferritin (Heald et al., 1979[link]), iron–sulfur clusters (Bunker & Stern, 1977[link]), the copper sites in oxy- and deoxy­hemocyanin (Brown et al., 1980[link]) and the nature of the vanadium metal complex in vanadocytes (Tullius et al., 1980[link]). Most of these early studies predate crystallographic structures and have stood the test of time, while others have since been superseded by new knowledge gained during the intervening period. Some forty years after the advent of the synchrotron era, structural biology is undergoing another revolution with the invention of the femtosecond pulsed X-ray free-electron laser (XFEL), which allows dynamic structures of biological molecules to be explored free of radiation damage (Spence & Chapman, 2014[link]). Recent advances in this field have demonstrated the potential to perform time-resolved L-edge XAS of metalloproteins (Mitzner et al., 2013[link]) and the value of combining room-temperature femtosecond crystallography with X-ray emission spectroscopy (XES; Kern, Hattne et al., 2014[link]). The latter approach has been used to investigate the water-oxidation mechanism of photosystem II (Kern et al., 2018[link]), adding a new chapter to a story that started in the early days of synchrotron BioXAS (Kirby et al., 1981[link]).

2. The relationship between macromolecular crystallography and XAS for biological systems

One of the main advantages of XAS as a structural technique compared with macromolecular crystallography (MX) is that crystals are not required: samples can be prepared and studied in any material state. This has a number of benefits for the structural determination of metal centres in proteins, as they can be probed under conditions that may not be easily accessible to MX, such as a selected pH or temperature, the binding of selected ligands, changes to metal oxidation states and physiologically more realistic in vivo sample environments. A major strength of XAS is that it is element-specific and generally provides more accurate (±0.02 Å or better) and precise metal–ligand bond lengths than MX (with an accuracy of approximately ±0.1 Å at 1.5 Å resolution)1. For example, a change in metal oxidation state may lead to subtle structural changes being well determined by changes to the XAS data (XANES or EXAFS) while remaining unobserved by MX due to insufficient resolution. This could lead to misidentification of the oxidation state in the protein crystal, as was seen for M148Q rusticyanin, where EXAFS detected a change in the Cu–S(Cys) distance of 0.05 Å upon reduction, a result that led to a reassignment of the oxidation state of the crystal structure (Barrett et al., 2006[link]). Metal oxidation states have not been experimentally determined in the majority of metalloprotein crystal structures in the Protein Data Bank, potentially leading to the misinterpretation of functional states. In addition to in situ Raman and single-crystal micro-spectrophotometry (Kekilli et al., 2017[link]), XANES can provide a way to interrogate redox and ligand states in crystals during MX data collection (Hough et al., 2008[link]).

BioXAS has several limitations, such as the generally weak scattering from light atoms, the often relatively short data ranges [typically ≤4 Å due to the increased Debye–Waller terms for more distant atoms and the decay of backscattering amplitudes with 1/(distance)2 or the presence of overlapping absorption edges], poor bond-length resolution for similar types of scattering atoms (typically ∼0.12 Å) and problems determining accurate coordination numbers or distinguishing between elements with similar atomic numbers (for example O and N). Atoms (ligands) seen by MX can under some circumstances be either be missed entirely by XAS, as in the case of the S(Met) ligand in the type 1 copper proteins, due to uncorrelated motions between the Cu and S atoms (Scott et al., 1982[link]), or can be difficult to determine because of out-of-phase interference of the backscattering from different atom types (Gu et al., 1996[link]). Optimum results can be obtained by careful experimentation, such as improving the bond-length resolution to <0.1 Å (Yano, Pushkar et al., 2005[link]), and through data analysis using the advanced software tools that are now available (see Part 6 of this volume), including the use of independent chemical information and crystal structure data. An early attempt to include chemical data from crystallo­graphy in XAS analysis was the imidazole rigid-body group fitting method of Co, Scott et al. (1981[link]). The inclusion of multiple scattering, enabling bond-angle determination in data analysis, then led to further advances (Strange et al., 1987[link]), which were first applied to CuZn superoxide dismutase (Blackburn et al., 1987[link]). Restrained and constrained fitting of EXAFS data in biological systems, analogous to the methods employed in crystallography, were introduced in 1992 (Binsted et al., 1992[link]) and were later extended to include fitting of the whole XAS spectrum (Binsted & Hasnain, 1996[link]). These developments, using metrical and geometrical data derived from small-molecule crystallography and exploiting the synergy between XAS and crystallography (Hasnain & Strange, 2003[link]; Strange et al., 2003[link]; Yano & Yachandra, 2009[link]; Cotelesage et al., 2012[link]), have become the standard approach for BioXAS and allow crystallographic information, including three-dimensional modelling, to be routinely and easily incorporated into XAS data analysis.

For biological materials, a serious issue for both XAS and (more so) MX using synchrotron radiation (SR) is the damage caused to the sample by the X-rays, with damage to protein crystals being proportional to the absorbed dose (Sliz et al., 2003[link]). To minimize the damage, experiments are therefore usually conducted by cryocooling samples to nonphysiological (liquid-nitrogen or liquid-helium) temperatures (Corbett et al., 2007[link]), thereby slowing the diffusion of X-ray-generated free radicals or hydrated electrons2. In XAS, the data range and signal to noise are also improved at liquid-nitrogen/helium temperatures. For MX, specific alterations to protein structure, such as the cleavage of disulfide bridges and the loss of carboxyl groups of acidic residues (Weik et al., 2000[link]), as well as a gradual loss of diffraction power and resolution, are the main observable effects. For BioXAS applications, photoreduction at metal centres has long been recognized as a major problem, especially for redox-sensitive metalloproteins (Chance et al., 1980[link]). This kind of damage may occur in protein crystals at much lower X-ray doses than are required to complete MX data collection, as shown for example for the Mn4Ca complex of photosystem II (Yano, Kern et al., 2005[link]; Grabolle et al., 2006[link]), leading to uncertainties in the interpretation and reliability of the final crystal structures. Photoreduction may result in changes to both the oxidation state and the coordination of a metal, which are key observables in any XAS study, and careful control and monitoring of a sample (a crystal or frozen solution) is therefore essential, for example by examining consecutive absorption-edge scans or by in situ optical spectrophotometry of the sample or off-line EPR. Time-resolved SR-based BioXAS experiments have successfully been conducted on radiation-sensitive samples using stopped-flow and freeze–quench methods to isolate selected functional states that are generally inaccessible to crystallo­graphy. For example, the ferryl radical compound I states that enable oxygen and C—H bond activation in P450 and chloroperoxidase have been determined in this way (Krest et al., 2015[link]).

3. Selected metalloprotein examples

The XAS literature related to biological systems is vast and it is not possible to do full justice to the wide range of its applications in this short review. A selected number of examples of metalloprotein systems of key biological interest have therefore been chosen to illustrate the insights into structure and function gained by XAS in relation to crystallography.

3.1. Photosystem II

The reaction centre of photosystem II (PSII), the water-splitting oxygen-evolving complex (OEC) in photosynthetic organisms, has been subjected to extensive XAS studies from the early days of SR-based X-ray spectroscopy to the present XFEL era. During this period, XAS has proved to be critical to the interpretation and the refinement of the associated crystallo­graphic data and structural modelling of the OEC (Yano et al., 2006[link]; Grundmeier & Dau, 2012[link]), which is now know to consist of an inorganic CaMn4O5 cluster with the Mn atoms linked by μ-oxo bridges and coordinated to amino-acid residues (Fig. 1[link]a). Four successive photons are required to oxidize one water molecule as the system cycles round the S0 → S1 → S2 → S3 → S4 (→ S0 with O2 release) states. These states have all been probed by static and time-resolved XAS experiments at cryogenic and room temperatures to reveal details of the water-splitting mechanism (Dau et al., 2001[link]; Yachandra et al., 1993[link]; Liang et al., 2000[link]; Messinger et al., 2001[link]; Haumann, Liebisch et al., 2005[link]; Haumann, Müller et al., 2005[link]). Mn K-edge EXAFS provided the first structural evidence for the existence of a μ-oxo-bridged metal cluster in the OEC (Kirby et al., 1981[link]), while XANES suggested a mixed metal valence state for the cluster, which is altered during the S1 → S2 transition (from Mn3+/Mn3+ to Mn3+/Mn4+) while the overall coordination is unchanged (Yachandra et al., 1987[link]). The average Mn–Mn distance in the cluster was determined to be ∼2.7 Å, with each Mn coordinated to three O/N atoms at ∼2 Å in addition to the two bridging O/N atoms and having 0.5–2 Mn neighbours. A shell of Mn or Ca atoms at ∼3.5 Å was also detected by XAS and suggested to form part of the cluster (MacLachlan et al., 1992[link]), and was subsequently confirmed by Ca K-edge EXAFS experiments (Cinco et al., 2002[link]) and anomalous diffraction on PSII crystals (Ferreira et al., 2004[link]). The metrical data from XAS, including the geometrical constraints specified by polarized EXAFS studies of oriented chloroplast membranes (George, Prince & Cramer, 1989[link]; Yachandra et al., 1993[link]) and single crystals (Yano et al., 2006[link]), provided crucial information on the composition, shape and size of the cluster prior to high-resolution crystallographic data becoming available. The first high (1.9 Å) resolution crystal structure, ostensibly of the S1 state of OEC, appeared in 2011 (Umena et al., 2011[link]), and for the first time resolved the positions of the metal atoms, bridging O atoms and protein ligands. Radiation damage to the OEC is a problem for SR-based crystallographic data collection (Yano, Kern et al., 2005[link]) and the metal cluster in this structure is likely to be in a partially reduced state (some Mn3+/Mn4+ converted to Mn2+ and longer Mn–Mn bonds than seen in EXAFS) rather than pure S1. Nevertheless, this structure provided a foundation for exploring the nature of the different S states of the catalytic cycle when used in combination with XAS data (Grundmeier & Dau, 2012[link]) and density-functional theory (DFT) modelling (Siegbahn, 2013[link]). Radiation-damage-free structures of PSII in different states and at varying resolutions (2.2–6.5 Å) have recently been reported using femtosecond XFEL crystallo­graphy (see, for example, Kern et al., 2012[link], 2018[link]; Kupitz et al., 2014[link]; Suga et al., 2019[link]; Sauter et al., 2016[link]). The quest to finalize the OEC structure and definitively establish the water-splitting mechanism therefore continues into the XFEL era, with XAS (and XES) playing a central role alongside crystallo­graphy.

[Figure 1]

Figure 1

A selection of metal centres in proteins that have been elucidated by XAS in conjunction with crystallographic studies. (a) The structure of the oxygen-evolving CaMn4O5 complex of photosystem II. (b) The structure of the FeMo cofactor of Mo-nitrogenase. S and Fe atoms are shown as yellow and orange spheres, respectively. (c) The active site of CuZn superoxide dismutase in its oxidized water-bound state. (d) The hydrogenase enzyme bimetallic Ni–Fe active-site cluster.

3.2. Nitrogenase proteins

XAS has been used to characterize the metal centres in several molybdenum-containing proteins or enzymes, including the nitrogenases, which catalyse a central reaction of the global nitrogen cycle: the fixing and reduction of N2 gas to NH3 with the associated release of H2. Nitrogenases are formed of two component metalloproteins, an Fe protein and an MoFe protein, which contains two FeMo cofactors (`FeMoco') and two FeS (`P') clusters. Alternative nitrogenases contain vanadium or iron in place of molybdenum. Nitrogenase was the first enzyme to be studied by XAS (Cramer et al., 1976[link]), and Mo K-edge XAS of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenases provided the first structural evidence for the presence of a common but unique Mo–Fe–S cluster in the MoFe protein, with 3–4 S atoms at ∼2.3 Å, 2–3 Fe atoms at ∼2.7 Å and one S atom at ∼2.5 Å (Cramer, Gillum et al., 1978[link]). V and Fe K-edge studies of the VFe protein of A. chroococcum nitrogenase demonstrated a structure analogous to FeMoco and suggested that 3–4 O/N atoms were coordinated to vanadium at ∼2.1 Å, along with two S atoms at ∼2.3 Å and three Fe atoms at ∼2.7 Å (Arber et al., 1987[link]). XAS was also used to propose the existence of an FeFe cofactor in the Fe-only nitrogenase from Rhodobacter capsulatus (Krahn et al., 2002[link]). While many of the structural models of FeMoco and the P-cluster proposed on the basis of XAS studies of the enzymes and synthetic Mo–Fe–S complexes were discarded following the publication of the first 2.7 Å resolution crystal structure of A. vinelandii nitrogenase (Kirn & Rees, 1992[link]), XAS proved its utility in providing structural constraints and in aiding interpretation of the crystallo­graphic data for the metal centres of the MoFe protein. The crystal structures showed [MoFe3S3] and [Fe4S3] clusters linked by three S2− bridges, with the molybdenum in the cluster directly coordinated to three S atoms, two O atoms from homocitrate and one N atom from a His ligand (Fig. 1[link]b). In the presence of the heavy atoms the homocitrate and histidine ligands in the cluster were less well determined in the initial crystal structures. A 3D EXAFS refinement (Cheung et al., 2000[link]) using 1.6 Å resolution crystallographic coordinates of the Klebsiella pneumoniae MoFe protein showed that EXAFS-derived distance restraints can be used in the early stages of crystallographic refinement to substantially improve the final crystallographic model (Strange et al., 2003[link]). The accuracy of this approach was confirmed by a 1.16 Å resolution crystal structure of the MoFe protein (Einsle et al., 2002[link]), which also revealed the presence of an interstitial light atom, later confirmed to be a C atom, coordinated by Fe at the centre of FeMoco. An analogous carbide at the centre of the FeV cofactor was confirmed by DFT modelling and XES data (Rees et al., 2015[link]). XAS and spectroscopic studies of nitrogenases have also focused on substrate binding and the probing of associated conformational changes in FeMoco. Conformational changes were observed to a long Fe—Fe bond length in FeMoco upon the binding of propargyl alcohol to an Fe atom adjacent to the Mo atom (George et al., 2012[link]), while EXAFS of the resting enzyme showed that the interaction between the Mo atom and the shell of Fe atoms originally at 5.08 Å was altered by reaction with CO (Scott et al., 2014[link]). Recently, XAS and crystallography were combined to assign individual Fe-atom oxidation states to the resting state of FeMoco (Spatzal et al., 2016[link]), while high-energy resolution fluorescence-detected XAS and EXAFS were used to probe the local electronic and geometric structure of selenium-substituted FeMoco to reveal antiferromagnetic coupling in the cluster (Henthorn et al., 2019[link]). The MoFe protein crystal structure has now been resolved to 1 Å resolution (Spatzal et al., 2011[link]), yet these recent experiments demonstrate that nearly 45 years after its first application and when used in combination with crystallography or other methods, for example vibrational spectroscopy and DFT, XAS still has an important role in discovering the important functional states of FeMoco in nitrogenase proteins.

3.3. Copper proteins

The redox cycling and structure of the copper and zinc centres in CuZn superoxide dismutase (SOD), which catalyses the conversion of the [{\rm O}_{2}^{{\bf \bullet}-}] radical to O2 and H2O2, has been explored by a number of XAS studies (Blackburn et al., 1987[link]; Murphy et al., 1997[link]); the first was by Blumberg et al. (1978[link]), who used Cu and Zn K-edge XANES to probe the electronic states of oxidized and reduced protein. The crystal structure of oxidized (bovine) SOD showed that four histidine ligands are coordinated to the solvent-exposed copper, with one of these histidine ligands being coordinated to zinc (Fig. 1[link]c). The loss of this Cu–His–Zn bridge and proton donation from the bridging histidine were proposed to be crucial steps in the catalytic mechanism (Tainer et al., 1983[link]). The first direct structural evidence for a three-coordinate copper site in reduced SOD was provided by Cu K-edge EXAFS (Blackburn et al., 1984[link]), with Zn K-edge EXAFS showing the zinc site to be unperturbed by the cycling of the redox state of copper. The first crystal structures of reduced SOD were ambiguous, suggesting an intact Cu+–His–Zn2+ bridge in reduced bovine SOD1 protein (Rypniewski et al., 1995[link]). A subsequent 1.15 Å resolution structure of fully reduced bovine SOD was shown to be consistent with the XAS data and the accepted catalytic mechanism (Hough & Hasnain, 2003[link]).

Type I (`blue') copper centres mediate electron-transfer reactions. Ligand K-edge XAS was used as a direct probe of the covalency of metal–ligand bonds (Glaser et al., 2000[link]). Experiments performed at the S K edge showed that the high covalency of the unusually short Cu—S(Cys) bond is responsible for the small hyperfine splitting observed in the type I proteins (Shadle et al., 1993[link]). The first direct structural information on the type I copper proteins was provided by Cu K-edge XAS of oxidized Pseudomonas aeruginosa azurin in 1978 (Tullius et al., 1978[link]). This study revealed the presence of a remarkably short (2.1 Å) Cu—S bond together with two or three N atoms coordinated at 1.97 Å. The first crystal structures were reported in 1978 for P. aeruginosa azurin (at 3 Å resolution) and Populus nigra plastocyanin (at 2.7 Å resolution), and confirmed that the active site consisted of two N(His) ligands, one S(Cys) ligand and one S(Met) ligand. EXAFS and other spectroscopic data showed that the hydrophobicity of the axial ligand is the dominant factor in determining the redox properties of type I proteins (Berry et al., 2003[link]). The function of the methionine ligand (or an alternative fourth ligand) in modulating the redox potential was also addressed by variable-pH spectroscopy, ligand binding and XAS of azurin mutants (Murphy et al., 1993[link]; Strange et al., 1996[link]) to show that the methionine ligand in the native protein constrains the active site to adopt a preferred Cu2+ orientation that facilitates conversion to Cu+. XAS studies of Rhus vernicefera stellacyanin conducted at different pH values showed that the fourth ligand was either N- or O-donating, at 2.7 Å, and not a sulfur ligand (Strange et al., 1995[link]). These findings from XAS were confirmed by the subsequent crystal structures, which showed that glutamine is the fourth ligand, with a Cu–O(Gln) distance of 2.2–2.5 Å (Hart et al., 1996[link]; Koch et al., 2005[link]).

3.3.1. Multi-copper centres

The type III copper site in hemocyanin was structurally characterized by EXAFS (Co, Hodgson et al., 1981[link]; Woolery et al., 1984[link]) in the decade before the first low (3.2 Å) resolution crystal structure was reported (Volbeda & Hol, 1989[link]). Subsequent improvements in resolution confirmed the EXAFS model of the copper coordination in the oxy (three histidine ligands and Cu atoms bridged by O atoms) and deoxy (three histidine ligands) forms of the protein. Multi-copper oxidases utilize a combination of type I, type II and type III copper centres to catalytically reduce O2 to H2O. Several years before the first crystal structures became available, EXAFS was used to show that a type II/type III trinuclear site in laccase is the minimal active site for this reaction (Cole et al., 1990[link]). Later, in combination with EPR and MCD spectroscopy, EXAFS was used to characterize reaction intermediates formed at the trinuclear centre during catalysis (Lee et al., 2002[link]). The mixed-valence binuclear CuA sites in cytochrome c oxidase and nitrous oxide reductase were both shown by EXAFS to have a very short (∼2.5 Å) Cu–Cu separation and each Cu atom was shown to be coordinated by S and light (N, O) atoms, including at least one histidine (Blackburn et al., 1994[link]; Charnock et al., 2000[link]).

3.4. Iron proteins

Iron–sulfur electron-transport proteins were early subjects of XAS studies. EXAFS was used to correct the initial crystal structure of rubredoxin, where an unusually short Fe—S bond in the single FeS(Cys)4 cluster had been proposed (Bunker & Stern, 1977[link]). Improved resolution (1.2 Å) crystal structures subsequently confirmed the EXAFS structural model. A systematic EXAFS study was also made of 2Fe and 4Fe iron–sulfur clusters in proteins and model compounds (Teo et al., 1979[link]), while the presence of an Fe3S4 site in the 3Fe iron–sulfur clusters in ferredoxin II (Antonio et al., 1982[link]) and the inactive form of aconitase (Beinert et al., 1983[link]) was established by EXAFS and was subsequently confirmed by crystallo­graphy (Kissinger et al., 1989[link]; Robbins & Stout, 1989[link]).

XAS studies of FeFe and NiFe hydrogenases, enzymes that perform the reversible oxidation of H2, are greatly complicated by the presence of different iron sites, i.e. in the multiple Fe–S clusters and in the active sites. Nevertheless, Ni K-edge EXAFS was used more than 30 years ago to demonstrate the presence of a novel Ni–S cluster in the active sites of the NiFe hydrogenases from Methanobacterium thermoautotrophicum (Lindahl et al., 1984[link]) and Desulfovibrio gigas (Scott et al., 1984[link]). In the FeFe hydrogenase from Clostridium pasteurianum, EXAFS showed the active site to contain an Fe–Fe interaction in the oxidized state at a distance of ∼2.8 Å, which increased by ∼0.5 Å upon reduction (George, Prince, Stockley et al., 1989[link]). While incomplete [for example, the ∼2.7 Å Ni–Fe interaction in the first crystal structure of oxidized D. gigas hydrogenase (Volbeda et al., 1996[link]) was not detected by EXAFS, possibly due to interference between Ni–Fe and Ni–S scattering (Gu et al., 1996[link])], these EXAFS-derived models suggested new kinds of metal-centred biological structures. The Fe–Fe enzymes are now known to consist of a combination of 4Fe–4S and Fe–Fe domains, with distinctive nonprotein CO and CN ligands of iron in the latter domain being essential for biological activity (the NiFe enzyme active site is similar; Fig. 1[link]d). XAS has shown that the irreversible O2-induced degradation of the active site, which is a problem facing the development of robust `hydrogenase-based' biofuel cells or biomimetics, results from specific damage to the 4Fe–4S (but not the Fe–Fe) domains (Stripp et al., 2009[link]). Using more refined methods of analysis and improved data quality, current XAS investigations have shown good agreement with, and have aided in the interpretation of, crystallographic data for the hydrogenases (Gu et al., 2003[link]; Hiromoto et al., 2009[link]).

Currently (mid-2020) around 5400 iron haem structures have been deposited in the PDB. Of these entries, only ∼40 unique proteins (∼170 non-unique) are solved to a resolution (<1.2 Å) where the accuracy and precision of XAS at the metal site is matched by crystallography. Important questions addressed by XAS include the nature of the subtle structural changes that occur upon the binding of ligands to the Fe atom in the ferric and ferrous forms, the small (∼0.5 Å) displacement of Fe from the haem plane in deoxyhaemoglobin (Eisenberger et al., 1978[link]; Perutz et al., 1982[link]), which is seen as a key factor in ligand-binding affinity and cooperativity, and intermediates in peroxide chemistry in P450 and analogous enzymes (see, for example, Stone et al., 2005[link]; Krest et al., 2015[link]). Many of the earlier studies were performed before crystal structures became available and, more recently, when used in combination with MX data, XAS continues to probe transient states that are inaccessible to crystallography. Multiple-scattering XANES has been used to probe the structures of the deoxy forms of myoglobin and haemoglobin, while polarized single-protein crystal experiments have been used to characterize carboxymyoglobin (Della Longa et al., 1999[link]), cyanomyoglobin (Arcovito et al., 2007[link]) and the photoreduction of the iron in aquomet-myoglobin (Della Longa et al., 2003[link]). Other steady-state XAS studies have included those in combination with laser flash photolysis to examine the geminate rebinding of carbon monoxide to myoglobin at cryogenic temperatures (Chance et al., 1983[link]; Della Longa et al., 2001[link]). Thirty years ago a groundbreaking submillisecond time-resolved XANES study was made of carboxymyoglobin, finding a −3 eV shift in the absorption energy of the transient photolysed product compared with the ligand-bound protein (Mills et al., 1984[link]). X-ray absorption experiments with <100 ps resolution on third-generation synchrotrons have recently allowed structural interpretations of the Fe K-edge spectra of transient carboxymyoglobin and nitrosomyoglobin complexes, showing ligand dissociation and doming of the haem (Stickrath et al., 2013[link]; Silatani et al., 2015[link]). Time-resolved XAS of carboxymyoglobin performed using an XFEL has suggested that a rearrangement of the haem occurs within the first 70 fs of photolysis occurring, with iron moving out of the haem plane within 400 fs (Levantino et al., 2015[link]). Structural information from photoactivated transition-metal complexes has also been demonstrated at XFELs using femtosecond-resolution EXAFS (Britz et al., 2020[link]).

XAS remains a powerful synchrotron-based X-ray method. It yields detailed local structural and electronic information on biological samples in different states of matter and does not require, although it can use, samples with long-range order or single crystals. The metal environments of `uncrystallizable' metalloproteins can easily be studied in their chemically relevant states. When used in combination with crystallo­graphic information, XAS can also provide important information to help interpret or improve crystallographic models and identify metal redox states. Techniques for rapid data collection for both MX and XAS experiments at synchrotrons and at XFELs will lead to an expansion of time-resolved applications to study catalysis in situ using crystals or other states of matter.

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