Báo cáo khoa học: Cytoglobin conformations and disulfide bond formation pdf

Spectroscopic studies have shown that Cygb belongs to the class of hexacoordinated globins, for which the Keywords disulfides, globins, kinetics, ligand binding, light scattering Corresp

Christophe Lechauve1, Ce´dric Chauvierre1, Sylvia Dewilde2, Luc Moens2, Brian N Green3, Michael

C Marden1, Chantal Ce´lier1and Laurent Kiger1

1 Inserm U779, Universite´s Paris VI et XI, Le Kremlin-Biceˆtre, France

2 Department of Biomedical Sciences, University of Antwerp, Belgium

3 Waters MS Technologies Centre, Micromass UK Ltd., Altrincham, Cheshire, UK

Introduction

The vertebrate heme globin family has been extended

to include two new members, neuroglobin (Ngb) and

cytoglobin (Cygb), which differ in structure, tissue

distribution and function [1–3]

Cygb is expressed at varying concentrations in many

body tissues, such as connective tissue, fibroblasts and

neurons [3–5] The cellular localization of Cygb is

atypical; it is present in the cytoplasm of almost all cell

types; it is also found in the cytoplasm and nucleus of

neuronal cells [4,6] The physiological role of Cygb

remains unknown, although several roles have been

proposed, such as the protection of cells from

oxida-tive-related damage during ischemic reperfusion injury

following hypoxia [7,8], the metabolism of NO in

tis-sues [9], and collagen synthesis [4,10] It has also been

reported that Cygb plays a role in cancer as a tumor

suppressor gene; indeed, it was observed that the

pro-moter region of Cygb is hypermethylated and under-expressed in tumors [11]

Cygb is considered to be in a clade with vertebrate myoglobin (Mb) and shares about 30% amino acid sequence identity with Mb, implying a common evolu-tionary ancestry [3] Human Cygb is composed of 190 amino acids with the presence of extended N- and C-terminal regions of about 20 residues each The crys-tal structure of this globin is characteristic of the clas-sical three-over-three a-helical globin fold in the core region of each subunit, and the asymmetric unit of the crystals contains two molecules of Cygb [12,13] Cygb contains two exposed cysteine residues (Cys B2 and Cys E9), suggesting the possibility of inter or intra-molecular disulfide bridge formation

Spectroscopic studies have shown that Cygb belongs

to the class of hexacoordinated globins, for which the

Keywords

disulfides, globins, kinetics, ligand binding,

light scattering

Correspondence

L Kiger, INSERM – U779, 78 rue du

Ge´ne´ral Leclerc, Hoˆpital de Biceˆtre Bat.

Broca, Niveau 3, 94275 Le Kremlin Biceˆtre,

France

Fax: (33) 1 49 59 56 61

Tel: (33) 1 49 59 56 64

E-mail: Laurent.Kiger@inserm.fr

(Received 18 February 2010, revised 8 April

2010, accepted 13 April 2010)

doi:10.1111/j.1742-4658.2010.07686.x

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form Thus, rather than displaying a compact globular form, cytoglobin behaves hydro-dynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads

to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude

Abbreviations

Cygb, cytoglobin; DLS, dynamic light scattering; Mb, myoglobin; Ngb, neuroglobin; SEC-MALLS, size exclusion chromatography with multi-angle laser light scattering.

binding of an external gaseous ligand to heme requires

the dissociation of an internal protein residue (His E7);

both ligands are in competition for the distal heme

bind-ing site A biphasic pattern was observed, as expected

for a hexacoordinated model of competition between

two ligands for a single binding site [14] In this study,

we re-examined the structure–function relationships of

Cygb, as several questions have been raised concerning

the basic properties of Cygb, namely the role of the

disulfide bond in protein function and its involvement in

the tertiary and quaternary structures

Results and Discussion

Quaternary structure analysis of human Cygb by

size exclusion chromatography with multi-angle

laser light scattering (SEC-MALLS) and DLS

The theoretical molar mass of Cygb is 21 404 Da Cygb

has been characterized previously by SEC as a dimeric

protein [12,14] The elution volume measured by SEC is

directly related to the hydrodynamic radius of the

pro-teins, and is generally correlated with the logarithm of

the molar mass The Cygb multimeric state was initially

determined using molar mass markers belonging to the

globin protein family, but this established method

depends on the reliability of the size of the markers and

may not be accurate in the absolute molar mass

deter-mination For this reason, we used static light

scatter-ing (MALLS) for the determination of the molar

masses of the different Cygb protein species in solution

SEC-MALLS analysis gave a molar mass of

21 400 ± 100 Da for Cygb in solution (Fig 1A) It

should be noted that this method does not rely on

reference proteins

We also estimated the hydrodynamic diameter using

two independent instruments (Malvern Instruments,

Malvern, Worcestershire, UK and Wyatt Technology,

Santa Barbara, CA, USA); it was found to be

indepen-dent of the redox and ligation states of Cygb The

hydrodynamic diameter versus molar mass was plotted

for Cygb (5.2 nm), together with several globular

pro-teins (Fig 1B) It appears that this size parameter

value for Cygb does not correlate well with the linear

relationship found between the hydrodynamic diameter

and molar mass for other proteins belonging to the

globin family SEC confirmed that the N- and

C-termi-nal extensions of Cygb ( 20 residues each) are

responsible for a large increase in hydrodynamic

vol-ume for Cygb relative to a globular protein A

trun-cated Cygb of approximately 17 kDa, without the

extensions, exhibits similar hydrodynamic properties to

those of ‘classical’ globins (Fig S1)

These results explain the misinterpretation of the Cygb quaternary assembly arising from gel filtration experiments based only on a putative relationship between the hydrodynamic volume and molar mass [12,14] It should also be noted that electrostatic repulsion (or attraction) forces between the proteins and column packing may contribute to a deviation from an ideal elution behavior based only on the hydrodynamic diameter, in this case for globular pro-teins The N- and C-terminal residue segments of Cygb were not clearly resolved in two of the X-ray crystal structures [12,13] In another structure display-ing covalently bound dimer through two intermolecu-lar disulfide bridges [15], the extensions formed an additional a-helix and an ordered loop, respectively,

A

B

Fig 1 (A) Molar mass determination of Cygb using SEC-MALLS: the thin full curve represents the relative UV absorbance at

280 nm; the bold line corresponds to the molar mass estimated from the light scattering profile and concentration measurement; both versus the elution time (B) Correlation between the hydrody-namic diameters (measured by dyhydrody-namic light scattering; Malvern Instruments and Wyatt Technology) and molar masses of six globu-lar proteins (monomeric a-globin, Ngb, Mb, tetrameric hemoglobin and octameric hemoglobin) The Cygb (red) hydrodynamic diameter deviates from the correlation line of other globin proteins.

but not in a symmetrical way, as only one extremity

is well defined in each subunit Thus, the

conforma-tional flexibility of both terminal regions may explain

the increase in hydrodynamic diameter relative to

other globins

Mass spectrometry assessment of Cygb disulfide

bonds and general structure

The results obtained by MALLS were complemented

by electrospray ionization mass spectrometry

(ESI-MS) When analyzed under conditions that should

retain the noncovalent bonds for globin assembly [14],

the dominant species was monomeric Cygb, together

with approximately 5% dimeric Cygb A solution of

Cygb with dithiothreitol, a thiol reducing agent,

ana-lyzed under denaturing conditions, showed

predomi-nantly a monomer with a mass of 21 404.7 ± 0.3 Da

(sequence mass 21 404.7 Da) and no dimer (Fig 2A)

When this solution was incubated at 37C and pH 8

after dithiothreitol removal, the mass of Cygb

progres-sively decreased over 7 h to 21 402.6 ± 0.3 Da

(Fig 2B), implying the formation of an intramolecular

disulfide bond (calculated mass 21 402.7 Da) Also

pres-ent was a minor componpres-ent (not shown) of mass

42 805.3 ± 5 Da ( 5% of Cygb), which is consistent

with a Cygb dimer formed by one or two intermolecular

disulfide bonds between two Cygb monomers (calculated masses of 42 807.3 and 42 805.3 Da, respec-tively) As expected, when the solution of Cygb used to produce Fig 2B was reduced with dithiothreitol, the mass of the Cygb monomer reverted to 21 404.7 ± 0.3 Da (Fig 2C), and the putative dimer disappeared These results confirm the occurrence of mainly mono-meric Cygb with an intramolecular disulfide bridge

Crystallographic structure The initial crystallographic structures of Cygb were described as dimers [12,13] There are major differences between these two structures One was obtained from

a mutated protein without cysteines [Cys38(B2)Ser and Cys83(E9)Ser mutant], whereas the other protein con-tained the native cysteines An asymmetric unit includ-ing two globins is observed for Cygb, and does not necessarily imply a dimeric form in dilute solutions However, Sugimoto’s dimeric structure (pdb 1V5H [13]), with two intermolecular disulfide bridges, is not present significantly in our results, which show mono-meric Cygb with an intramolecular disulfide bridge at protein concentrations of 0.1–20 lm Structure com-parison shows a difference at the position of Cys B2, with the distance between Ca atoms for residues B2 and E9 of 7.35 A˚ in de Sanctis’ structure (pdb 1UM0

Reduced Cygb

Oxidized Cygb

Reduced Cygb

A

B

C

Fig 2 ESI-MS analyzed under denaturing conditions of: (A) a solution of reduced Cygb; (B) solution in (A) after 7 h of incubation at 37 C under aerobic conditions, pH 8; (C) solution in (B) after reduction with dithiothreitol.

[12]) and 12.0 A˚ in Sugimoto’s structure (Fig 3) There

is a loss of one turn of the B-helix in Sugimoto’s

struc-ture, with Cys B2 moving away from the Cys E9

posi-tion, which would not allow an intramolecular

disulfide bridge (Fig 3) The buried interface surface

of Sugimoto’s structure [13] is half that (640 A˚2) found

for another Cygb dimeric structure (2DC3 [15])

exhib-iting the same intermolecular disulfide bonds In this

latter crystal structure, in addition to the fact that only

one terminal sequence was ordered in each monomer

(one a-helix and one ordered loop motif), structural

differences were also found in the conformation of

certain residues in the vicinity of heme

Ligand binding of human Cygb

Cygb shows the characteristic absorption spectra

(Fig S2) and ligand binding for the hexacoordinated

globins The kinetics of ligand binding to Cygb after

the photodissociation of CO shows the form expected

for hexacoordinated globins (Fig 4A) The rapid

bimolecular phase corresponds to the competitive

binding between CO and the internal histidine

residue (His E7) For the fraction binding histidine, the return to the final (CO-bound) state involves the slow dissociation of histidine A previous study [16] has reported a single exponential decay, probably because the observation time was not sufficiently long for histidine dissociation and replacement by CO

E9 B2

Fig 3 Two Cygb crystallographic structures, superimposed by the

full sequence method of DS-Visualizer (Accelrys), which takes into

account all common atoms of the aligned globins The residues

dis-played correspond to the position for the cysteine pair (B2 and E9)

and the distal and proximal histidines (E7 and F8) in Cygb The

structure in red of human Cygb was obtained by Sugimoto et al.

[13] (1V5H) and the structure in green (B2 and E9 in purple) was

obtained by De Sanctis et al [12] with a mutated protein (1UMO)

without cysteines: Cys38(B2)Ser and Cys83(E9)Ser.

B A

Fig 4 Flash photolysis kinetics for Cygb at 25 C (A) Recombina-tion kinetics at different CO concentraRecombina-tions [from top to bottom: 0.01, 0.1 and 1 atm CO (760 Torr)] After flash photolysis of CO, the first phase represents competitive binding between CO and histidine to the heme sites The second phase is a slow replace-ment reaction of histidine by CO to return to the preflash state The broken lines are simulations using the model for competitive ligand binding (B) Kinetic curves of the slow phase Samples with dithiothreitol (DTT)-reduced Cygb show a single form (also observed for the mutant Cygb without cysteines) The cysteine oxi-dized form, which can form an internal S–S bond, displays a faster replacement reaction; however, heterogeneity is observed, indicat-ing the presence of two protein conformations The rapid phase arising from the competition between CO and histidine for heme rebinding (with an observed rate equal to the sum k CO

on + k His

on ) is the same for samples with or without DTT.

By analyzing the data from experiments performed

at different CO concentrations, one can extract the

rates for CO and histidine association, as well as those

for histidine dissociation [14,17] Results from

experi-ments with a mixed CO–O2atmosphere allow a

deter-mination of the O2 binding rates The intrinsic O2 or

histidine dissociation is quite slow, requiring about 1 s

(Fig 4A)

Overall, the kinetics show the same form as for

Ngb, although the ligand association rates to Cygb are

slower than those to Ngb As this occurs for both

oxy-gen and histidine, the overall oxyoxy-gen affinity remains

on the order of 0.1–10 Torr at 25C, which is a

common range of O2 affinity measured for the

hexaco-ordinated globins For both Cygb and Ngb, the slow

oxygen and histidine dissociation rates compensate for

the effect on the overall oxygen affinity In the case of Ngb, there is a shift in the observed oxygen affinity from about 1 to 10 Torr on reduction of the cysteines; the internal mechanism is mainly caused by a change

in the histidine dissociation rate Cygb shows a similar effect, with the oxygen affinity changing from about 0.2 to 2 Torr (Fig 5); however, the transition only occurs for about one-half of the molecules

The major change in kinetics of Cygb after the addi-tion of dithiothreitol, known to reduce cysteines and therefore to break S–S bonds, is shown in Fig 4B Although the cysteine-reduced form shows homoge-neous kinetics, there is heterogeneity of the histidine dissociation rate for the protein solution exposed to O2 after purification Two histidine dissociation rates are measurable, each representing about one-half of the sample, and differ by a factor of 10 (Fig 4B, Table 1), whereas the other binding parameters are little changed

This heterogeneity could not be eliminated by vari-ous preparations from different laboratories or by changing the temperature and pH conditions It thus appears to be a basic property of Cygb samples One can then question whether there is an interconversion

of the two conformations Any interconversion of the conformations is apparently slow, as the ligand bind-ing and Fe2+ oxidation kinetics (which occur on the order of several minutes) display distinct phases for the two forms Indeed, a partial autoxidation under air

of the heme iron results in an enhancement of the higher oxygen affinity fraction (the more slowly oxidiz-ing form) as assessed by flash photolysis (data not shown)

The stopped-flow data for the replacement reaction indicate a further complexity For samples with and without dithiothreitol, at least two rates were observed (this work) [18] This could indicate several possible conformations for the distal histidine, and only certain

of these forms are apparent when the sample has been incubated in the His-Fe-His state As seen in the

P 50

0.1

1

10

100

Cygb S-S

Oxygen affinity

Cygb T-state HbA

Mb R-state HbA

Ngb S-S Ngb

Fig 5 Synthesis of the oxygen affinity for the various allosteric

states of members of the globin family For Ngb and Cygb, the

oxy-gen affinity, conserved for various species, falls within the

physio-logical range, supporting a role for oxygen delivery.

Table 1 Kinetic parameters determined from flash photolysis DTT, dithiothreitol; WT, wild-type.

Protein

k CO on

(l M Æs)1)

k O 2

on

(l M Æs)1)

kO2

off

(s)1)

K O 2 (n M )

k His on

(s)1)

k His off

(s)1) K His

PO2

50

(Torr)

Cytoglobin WT with S–S bridge

Experimental conditions: 50 m M phosphate buffer at pH 7.0, 25 C The O 2 solubility coefficient was taken as 1.82 l M ÆTorr)1 Note the distinction between the intrinsic affinity K O 2 = k off ⁄ k on (as for pentacoordinated forms) and the overall affinity observed for the ligand compe-tition of the hexacoordinated globins KO2 = (koff⁄ k on ) ⁄ (1 + K His ).

structural images (Fig 3), at least two distinct

posi-tions for Cys(B2)38 can be observed for Cygb without

the internal disulfide bond; this residue may thus adopt

various intermediate positions which may influence the

ligand binding kinetics

A slow relaxation process might explain the two

conformations as, in the stopped-flow experiments, the

protein is incubated in the His-Fe-His state for at least

several minutes It should be noted that no change in

the proportion of the kinetic phases was observed after

repeated photolysis of the hexacoordinated form over

5 s by a series of laser pulses at 10 Hz to remove the

CO ligand (for samples equilibrated under a 5% CO

atmosphere), meaning that slow relaxation between the

Cygb conformations does not take place within a

time-scale of seconds We also measured by stopped flow

the histidine replacement by CO, starting from the

hexacoordinated ferric form Using a double mixing

sequence, one can first reduce the iron and then

incu-bate Cygb in the ferrous hexacoordinated state for

var-ious delay times before mixing with CO to probe the

histidine to CO replacement reaction No difference

was observed for incubation times ranging from

milli-seconds to milli-seconds, meaning that the redox state of

iron does not influence the Cygb conformation in the

bis-histidyl bound state As the rapid phase of the

his-tidine to CO replacement reaction seems to be most

characteristic of the flash photolysis method, this

spe-cial state could depend on the recent history of binding

of an external ligand

For both Cygb and Ngb [14], the disulfide bond

influences the final position of the E-helix, which, in

turn, modifies the affinity for the distal histidine As

the overall O2 affinity depends on histidine binding,

this leads to a modification in O2 affinity The

mecha-nism in Ngb involves a disulfide bond in the CD loop

region which influences the position of the E-helix For

Cygb, a more direct mechanism can be envisaged from

the structure: the Cys E9 and the distal His E7 are on

opposite sides of the E-helix (Fig 3); thus formation

of the E9–B2 bond should pull directly on His E9

This change in conformation could also perturb the

local molecular dynamics, which may influence ligand

binding

Cygb presents the same features as Ngb; however,

the sample with S–S bonds is not homogeneous Only

about one-half of the signal corresponds to a high

dis-sociation rate for histidine and, consequently, one-half

to a low dissociation rate, the slow rate being similar

to that measured in the absence of the disulfide bridge

(Fig 4B) For both Ngb and Cygb, this transition is

reversible: reduction of the protein by dithiothreitol

leads to a single kinetic species, and the re-oxidation

of cysteines (to re-form the disulfide bridge) again exhibits heterogeneous kinetics

This reduction after the addition of 2 mm dithiothre-itol takes a few minutes at 37 C based on the CO binding kinetics, whereas S–S bridge formation under air is achieved after 7 h as measured by mass spectros-copy (Fig 2) Therefore, a faster formation of the disulfide bond, necessary for a physiological role, would probably require the presence of a catalyst

A catalyst of disulfide bridge formation that acts with

a coupling reaction between both redox centers, namely heme and cysteines, could also be considered Whatever the putative partner responsible for cysteine oxidation, the reverse reduction is probably performed

by glutathione The highly concentrated glutathione redox couple (GSH⁄ GSSG) is known to be displaced towards the reduced form in the cytosol, whereas, in the endoplasmic reticulum, most of the constitutive disulfide bridges are usually formed in secretory pro-teins Therefore, the equilibrium for disulfide bridge formation in Cygb will be influenced by the redox state

of the cytosol, which may also be transiently influ-enced by reactive oxygen species or other oxidative species derived from nitric oxide It should be noted that intracellular compartments may provide favorable local environments in which the thiol redox state may

be different from that of the bulk cytoplasm, but also that Cygb can be present elsewhere in the nucleus [6]

Conclusion

In conclusion, human Cygb is monomeric in solution,

as measured by MALLS, at micromolar concentrations

of heme, the range expected for Cygb in vivo The molar mass of Cygb samples measured by SEC-MALLS, mass spectrometry and flash photolysis con-firms the possibility of formation of an intramolecular disulfide bridge The formation of an intermolecular disulfide bridge was observed in mass spectrometry, but represents only a small fraction of the Cygb prepa-ration and is negligible in the kinetic analysis A con-formational transition in Cygb between an oxidized (intramolecular disulfide bond) form and a reduced (disulfide free) Cygb form is evident in the kinetics; such a transition affects the E-helix position, allowing

a fine modulation of the endogenous His E7 affinity for heme binding Disulfide bridge formation probably creates a significant stress on the E-helix of Cygb, resulting in a change in the distal histidine end-position

of the liganded and unliganded states, as well as in its movement for heme binding A constraint provided by the disulfide bridge creates two possible positions of distal histidine after photodissociation, leading to

heterogeneity of kinetics After breaking the S–S bond,

the histidine relaxes towards a stable conformation,

and kinetic curves show a clean biphasic form, as

expected for the competition model of two ligands with

one affinity for histidine It can be proposed, as for

Ngb [14], that, on oxidative stress, a change in the local

redox state in response to physiological stimuli may

induce the rupture and⁄ or formation of the

intramolec-ular B2–E9 disulfide bond in Cygb, thus initiating a

conformational change that affects the overall oxygen

affinity (Fig 5) and other sensing functions as well

Materials and methods

Expression and purification of recombinant Cygb

The expression of wild-type human Cygb and the doubly

mutated Cygb C38S⁄ C83S mutant was performed as

described previously [19] The truncated CYGB differs from

the wild-type by the removal of the amino-terminal residues

1–17 and the carboxyl-terminal residues 165–190, as

described previously [14] Inclusion bodies were solubilized

in 6 m guanidinium hydrochloride and, after elimination of

the insoluble material, Cygb was reconstructed by adding

free hemin and dialyzed overnight The samples were then

purified with an Akta purifier system (GE Healthcare,

Life-sciences, Uppsala, Sweden) on a Hitrap DEAE Sepharose

column (GE Healthcare, Lifesciences, Uppsala, Sweden)

The concentrated material was loaded onto a Superose 12

HR 16⁄ 50 column (GE Healthcare) The final purity of the

pooled Cygb was checked by absorbance spectra and

SDS-PAGE

Dynamic light scattering

The particle size was measured with a Zetasizer Nano-ZS

(Malvern Instruments), based on dynamic light scattering

Size distribution by volume was used for data

interpreta-tion Measurements were performed at 20C in 100 mm

NaCl, 30 mm phosphate buffer at pH 7.5, in triplicate on

each sample, and the average was taken for the diameter

Size exclusion by fast protein liquid

chromatography and MALLS

The Cygb shape and molar mass in solution were

deter-mined using online SEC-MALLS The gel filtration

separa-tion was carried out using an Ettan LC liquid

chromatography system (GE Healthcare) equipped with a

Superose 12 HR 10 ⁄ 300 GL column (GE Healthcare)

Isocratic elution was performed at a flow rate of

0.39 mLÆmin)1 using a mobile phase of 30 mm NaCl⁄ Pi

(pH 7.5), 150 mm NaCl and 0.03% sodium azide at 25C

It should be noted that NaCl was added to the elution

buf-fer to avoid unwanted interactions between proteins and the solid phase Light scattering analysis was performed using an Ettan LC HPLC system with automatic degasser and thermostatically controlled autosampler, connected inline to a DAWN HELEOS II 18-angle static light scattering detector, equipped with a QELS (quasi-elastic light scattering) instrument (Wyatt Technology) and an Optilab rEX differential refractometer, equipped with a Peltier temperature-regulated flow cell maintained at 25C (Wyatt Technology) Calibration of the light scattering detector was subsequently verified using an albumin mono-mer standard (Sigma-Aldrich, Copenhagen, Denmark), recombinant Ngb, Mb (Sigma-Aldrich, Copenhagen, Den-mark), Mb dimer, diaspirine cross-linked (DCL) hemoglo-bin tetramer (Baxter Healthcare Corporation, Deerfield, IL, USA) and an octameric recombinant hemoglobin based on the natural variant hemoglobin Ta-Li [20] The molar mass for the protein was calculated from the light scattering data using a specific refractive index increment (dn⁄ dc) value of 0.183 mLÆg)1 The light scattering on the different detectors was analyzed using astra v software (Wyatt Technology, version 5.3.4.13) to finally obtain the absolute molar mass and the hydrodynamic diameter for the eluted fractions

Sample preparation and ESI-MS

Native samples were infused (5 LÆmin)1) into the mass spec-trometer (Quattro Ultima, Micromass Ltd., Wythenshawe, UK) at a concentration of approximately 5 lm in 1 : 1 ace-tonitrile–water containing 0.2% formic acid Data were acquired over the mass-to-charge ratio (m⁄ z) range 600–

2000 (5 min) and deconvoluted to present the spectra on a molar mass scale using the maximum entropy (maxent)-based software supplied with the spectrometer Mass scale calibration employed the series of ions with multiple charges from separate introductions of Mb (sequence mass

16 951.5 Da)

Spectra and ligand binding⁄ dissociation kinetics

Spectral measurements were made with a Varian Cary 400 (Varian, Inc., Palo Alto, CA, USA) or an HP 8453 diode array spectrophotometer (Hewlett Packard, Bracknell, UK) All ligand binding experiments were performed in

30 mm phosphate buffer at pH 7.5 with (10 mm) or without dithiothreitol to reduce the cysteine residues

The binding kinetics after heme ligand photolysis were performed using an Nd:YAG laser CFR-300 (Quantel, Les Ulis, France) generating 8 ns pulses of 160 mJ at 532 nm The laser beam, as well as the monochromatic detection light, were brought to the sample cuvette by an optical fiber The methods used to assess the hexacoordination and bimolecu-lar CO and O2rate constants have been described previously [17] Samples from 1 to 10 lm on a heme basis were placed in

4 mm·10 mm quartz cuvettes We also used a SFM-3

stopped-flow rapid mixing equipment (Bio-logic SAS, Claix,

France) to study ligand replacement Experiments were

repeated at least three times for each sample condition

Acknowledgements

We thank Professors T Burmester (University of

Hamburg) and T Hankeln (University of Mainz) for

the Cygb plasmid, and Veronique Baudin-Creuza

(Inserm U779) for the octameric recombinant

hemo-globin sample This work was supported by Inserm,

DGA (De´le´gation Ge´ne´rale pour l’Armement) contract

N 07.34.004, and Universite´s Paris VI et XI

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Supporting information

The following supplementary material is available: Fig S1 Partition coefficients measured by gel filtration

Fig S2 Absorbance spectra of Cygb.

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