How protein structure affects redox reactivity: example of Human centrin 2.

Electron transfer inside proteins plays a central role in their reactivity and biological functions. Herein, we developed a combined approach by gamma radiolysis and electrochemistry which allowed a deep insight into the reactivity of Human centrin 2, a protein very sensitive to oxidative stress and involved in several key biological processes. This protein bears a single terminal tyrosine and was observed to be extremely sensitive to ionizing radiation sources, leading to a tyrosine dimer. By cyclic voltammetry in the 100-1000 V s(-1) range, its redox potential and dimerization rate could be evaluated. Accordingly, reaction in solution with a redox mediator revealed an efficient catalysis. Finally, protein denaturation by a progressive increase in temperature was proportional to a decrease of dimerization radiolytic yield. Our results thus demonstrated that the protein structure plays a major role in oxidation sensitivity. This leads to meaningful results to understand protein redox reactivity.

High-pressure SANS and fluorescence unfolding study of calmodulin.

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80°C, whereas the calcium-saturated form is stable up to temperatures as high as 100°C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000bar (300MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.

The E144 residue of Scherffelia dubia centrin discriminates between the DNA repair protein XPC and the centrosomal protein Sfi1.

Centrins are members of the EF-hand family of calcium-binding proteins, which are highly conserved among eukaryotes. Centrins bind to several cellular targets, through a hydrophobic triad. However, the W(1)xxL(4)xxxL(8) triad in XPC (Xeroderma Pigmentosum Group C protein) is found in the reverse orientation, as in the L(8)xxxL(4)xxW(1) triad in Sfi1 (Suppressor of Fermentation-Induced loss of stress resistance protein 1). As shown by previous NMR studies of human centrin 2 in complex with XPC or Sfi1, the E148 residue of human centrin 2 is in contact with XPC but is pushed away from the triad of Sfi1. We corroborated these findings using site-directed mutagenesis to generate mutations in Scherffelia dubia centrin (SdCen) and by using isothermal titration calorimetry to analyze the binding affinity of these mutants to XPC and Sfi1. We mutated the F109 residue, which is the main residue involved in target binding regardless of triad orientation, and the E144 residue, which was thought to be involved only in XPC binding. The F109L mutation reduced the binding of SdCen to XPC and Sfi1 and the negative effect was greater upon temperature increase. By contrast, the E144A mutation reduced the binding to XPC but had no effect on Sfi1 binding. The F109L-E144A mutation enhanced the negative effect of the two single mutations on XPC binding. Sfi1 proteins from Ostreococcus lucimarinus and Ostreococcus tauri, which belong to the same clade as S. dubia, were also investigated. A comparative analysis shows that the triad residues are more conserved than those in human Sfi1.

Probing structural and motional features of the C-terminal part of the Human Centrin 2/P17-XPC microcrystalline complex by solid-state NMR spectroscopy.

Insight into structural and motional features of the C-terminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine the quality of solid-state NMR spectra and the internal mobility of the protein. Two-dimensional (2D) (13)C-(13)C and (15)N-(15)N correlation spectra reveal intra- and inter-residue dipolar connectivities and provide partial, site-specific assignments of (13)C and (15)N resonance signals. The secondary structure of the C-ter HsCen2/P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca(2+) cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC.

Oxidative stress induces mainly human centrin 2 polymerisation.

PURPOSE: To determine the human centrin 2 (Hscen 2) protein response to oxidising radicals in vitro and to evaluate the consequences on its biological functions. MATERIALS AND METHODS: Hscen 2 was submitted to hydroxyl and azide radicals produced by radiolysis in the absence of oxygen. The resulting products were characterised by biochemical, spectroscopic and mass spectrometry techniques. Their thermodynamics parameters of complexation with C-terminal fragment of Xeroderma pigmentosum C protein (C-XPC), one of the Hscen 2 cellular partners, were quantified by isothermal titration calorimetry (ITC). RESULTS: Both hydroxyl and azide radicals induce centrin 2 polymerisation as we characterised several intermolecular cross-links generating dimers, trimers, tetramers and higher molecular mass species. These cross-links result from the formation of a covalent bond between the only tyrosine residue (Tyr 172) located in the C-terminal region of each monomer. Remarkably, dimerisation occurs for doses as low as a few grays. Moreover, this Hscen2 dimer has a lower affinity and stoechiometry binding to C-XPC. CONCLUSIONS: These results show that as oxidative radicals induce high proportions of irreversible damages (polymerisation) centrin 2 is highly sensitive to ionising radiation. This could have important consequences on its biological functions.

Scherffelia dubia centrin exhibits a specific mechanism for Ca(2+)-controlled target binding.

Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.

Structure, dynamics and thermodynamics of the human centrin 2/hSfi1 complex.

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the approximately 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide. The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -A translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C-HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III, made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand.

The carboxy-terminal domain of xeroderma pigmentosum complementation group C protein, involved in TFIIH and centrin binding, is highly disordered.

Xeroderma pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction. Here, we present the molecular and structural characterization of a C-terminal fragment of XPC (C-XPC: 126 residues, 815-940), which was shown to be involved in centrin 2 and TFIIH binding. C-XPC may be highly expressed in E. coli, but because of its limited solubility it was purified under 6 M urea. Using bioinformatics tools, and a combination of several experimental methods (circular dichroism, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering), we show that C-XPC has a highly flexible structure under native physiological conditions, with a propensity to form helical secondary structures. Isothermal titration calorimetry experiments show that the C-XPC fragment binds human centrin 2 with high affinity and a 1:1 stoichiometry. NMR analysis indicates that the physical interaction between C-XPC and centrin 2 induces only minor conformational changes into XPC, localized around the 17-mer segment (847-863), showed to be critically involved in the centrin binding.

Biophysical study of thermal denaturation of apo-calmodulin: dynamics of native and unfolded states.

Apo-calmodulin, a small, mainly alpha, soluble protein is a calcium-dependent protein activator. This article presents a study of internal dynamics of native and thermal unfolded apo-calmodulin, using quasi-elastic neutron scattering. This technique can probe protein internal dynamics in the picosecond timescale and in the nanometer length-scale. It appears that a dynamical transition is associated with thermal denaturation of apo-calmodulin. This dynamical transition goes together with a decrease of the confinement of hydrogen atoms, a decrease of immobile protons proportion and an increase of dynamical heterogeneity. The comparison of native and unfolded states dynamics suggests that the dynamics of protein atoms is more influenced by their distance to the backbone than by their solvent exposure.

Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein.

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

High sensitivity of human centrin 2 toward radiolytical oxidation: C-terminal tyrosinyl residue as the main target.

Centrins are calcium-binding proteins that play a significant role in the maintenance of the centrosomal organization, mainly in the continuity between centrosome and microtubular network. Recent data showed that centrosome duplication abnormalities, like overduplication for example, could be due to hydrogen peroxide, suggesting an important impact of oxidative stress. To challenge this hypothesis, we performed one-electron oxidation experiments with human centrin 2, starting from azide radicals. Our results first revealed several intermolecular cross-links generating dimers, tetramers, hexamers, and higher molecular mass species. Dimers result from covalent bond linking the C-terminal tyrosines of each monomer. Second, the methionyl residue at position 19 was oxidized on the monomeric centrin. Further, electron microscopy experiments on centrin 2 showed a preexisting hexameric organization that was stabilized by covalent bonds as a result of irradiation. Overall, these results show that centrin 2 is highly sensitive to ionizing radiation, which could have important consequences on its biological functions.

Slow backbone dynamics of the C-terminal fragment of human centrin 2 in complex with a target peptide probed by cross-correlated relaxation in multiple-quantum NMR spectroscopy.

The C-terminal domain of human centrin 2 (C-HsCen2) strongly binds to P1-XPC, a peptide comprising 17 amino acids with a NWKLLAKGLLIRERLKR sequence. This peptide corresponds to residues N847-R863 of XPC, a protein involved in the recognition of damaged DNA during the initial step of the nucleotide excision repair pathway. The slow internal dynamics of the protein backbone in the C-HsCen-P1-XPC complex was studied by measuring the relaxation rates of zero- and double-quantum coherences involving neighboring pairs of carbonyl 13C and amide 15N nuclei. These relaxation rates, which reflect dynamics on time scales in the range of micro- to milliseconds, vary significantly along the protein backbone. Analysis of the relaxation rates at different CaCl2 concentrations and ionic strengths shows that these slow motions are mainly affected by the binding of a Ca2+ ion to the lower-affinity EF-hand III. Moreover, we discuss the possible functional role of residues that undergo differential exchange in the formation of HsCen homodimers.

Binding of human centrin 2 to the centrosomal protein hSfi1.

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.

Crystallization and preliminary X-ray diffraction data of the complex between human centrin 2 and a peptide from the protein XPC.

Centrins are highly conserved calcium-binding proteins involved in the nucleotide-excision repair pathway as a subunit of the heterotrimer including the XPC and hHR23B proteins. A complex formed by a Ca2+-bound human centrin 2 construct (the wild type lacking the first 25 amino acids) with a 17-mer peptide derived from the XPC sequence (residues Asn847-Arg863) was crystallized. Data were collected to 1.65 angstroms resolution from crystals grown in 30% monomethyl polyethylene glycol (MPEG) 500, 100 mM NaCl and 100 mM Bicine pH 9.0. Crystals are monoclinic and belong to space group C2, with two molecules in the asymmetric unit. The unit-cell parameters are a = 60.28, b = 59.42, c = 105.14 angstroms, alpha = gamma = 90, beta = 94.67 degrees. A heavy-atom derivative was obtained by co-crystallization with Sr2+. The substitution was rationalized by calorimetry experiments, which indicate a binding constant for Sr2+ of 4.0 x 10(4) M(-1).

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair.

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly.

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.

Calcium and magnesium binding to human centrin 3 and interaction with target peptides.

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.

Disentangling ligand migration and heme pocket relaxation in cytochrome P450cam.

In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of carbon monoxide rebinding with cytochrome P450cam display up to five distinct processes between 77 K and 300 K. They were disentangled by using a combination of three approaches: 1), the competition of the ligand with xenon for the occupation of internal protein cavities; 2), the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; and 3), molecular mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek internal cavities. In cytochrome P450cam, active site conformational relaxation results from the displacement of the substrate toward the heme center upon photodissociation of the ligand. It is responsible for the long, puzzling bimodal nature of the rebinding kinetics observed down to 77 K. The relaxation rate is strongly substrate-dependent. Ligand migration is slower and is observed only above 135 K. Migration and return rates are independent of the substrate.

Calcium-dependent self-assembly of human centrin 2.

Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca(2+)-sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (Delta25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 x 10(5) m(-1)) to preformed oligomers of Delta25HsCen2 through an entropy-driven mechanism.

Competition with xenon elicits ligand migration and escape pathways in myoglobin.

Evidence for ligand migration toward the xenon-binding cavities in myoglobin comes from a number of laser photolysis studies of MbO2 including mutants and from cryo- and time-resolved crystallography of MbCO. To explore ligand migration in greater detail, we investigated the rebinding kinetics of both MbO2 and MbCO under a xenon partial pressure ranging from 1 to 16 atm over the temperature range (293-77 K). Below 180 K xenon affects to a significant, but minor, extent the thermodynamic parameters for rebinding from the primary docking site in each Mb taxonomic substate. Above 200 K the ligand migrates to the proximal Xe1 site but when the latter is occupied by xenon a new kinetic process appears. It is attributed to rebinding from transient docking sites located on the path between the primary and the secondary docking site of both ligands. Ligand escape exhibits a more complicated pattern than expected. At room temperature O2 and CO escape appears to take place exclusively from the primary site. In contrast, at T approximately 250 K, roughly 50% of the CO molecules that have escaped from the protein originate from the Xe1 secondary site.