Mem. Grad. School Sci. & Technol., Kobe Univ., 22-A; 21-31 (2004).

Photoionization and Photodissociation Processes of MultiplyCharged Cytochrome c Ions Produced by Electrospray Ionization
Shinji NONOSE, Hiroaki TANAKA, and Kiyokazu FUKE
Department of Chemistry, Faculty of Science, Kobe University
Abstract
Photo-induced reactions of multiply-charged cytochrome c ions, (M + nH)n+ (n = 9 – 17) areinvestigated at 28200 and 18800 cm-1 by using a tandem mass spectrometer with electrosprayionization source. Photoionization is found to be the dominant process for the lower charged states (n= 9 – 12) and its yield decrease rapidly with increasing the charge. The photoionization is ascribed tothe emission of electron by multiphoton excitation of heme under the influence of Coulomb attractive
potential arising from the charges in the polypeptide chain. Model calculations of the Coulomb
potential suggest that the structure of the polypeptide chain is completely elongated.
Key Words: Electrospray ionization; Photoionization; Cytochrome c ions; Coulomb potential;
Conformations of protein ions

1. Introduction
The structures and reactions of gas-phase clusters have been studied extensively using various
spectroscopic methods in order to bridge the gap between the gas phase and the condensed phase [1-4].
For examples, our group and Schulz’s group have investigated the electronic structures of the clusters
such as metal atoms and/or ions solvated with polar molecules for modeling microscopic solvation [512]. Recent advances in electrospray ionization (ESI) allow us to produce various kinds of nonvolatile
molecules in the gas phase without destruction [13,14]. The ESI mass spectrometry is now routinely
used for sensitive analytical determination of biological molecules, accurate measurement ofmolecular weights and sequence information. Several groups have applied ESI to the spectroscopic
studies of gas-phase cluster ions [15-18]. These studies provide information on the microscopic
solvation and the reaction dynamics of gas-phase clusters.
The conformations of large biological molecules such as proteins in the condensed phase arestrongly influenced by surrounding solvent environments. In order to investigate the intra- andintermolecular interactions, nonvolatile molecules should be introduced into the gas phase as ions. The
gas phase is an unusual environment for the investigation of biological molecules. However, the gasphase studies are expected to provide a deeper understanding of the intramolecular interactions as wellas hydration interactions that determine the conformations. Several different techniques have been
employed to obtain information about the conformations of large biological molecules in the gas phase.
McLafferty, Williams, and their collaborators have examined electron-capture kinetics [19], H/D
exchange kinetics [20], proton-transfer kinetics [21], or blackbody infrared radiative dissociation [22].

Jarrold and coworkers have performed ion mobility measurements to study the geometries of naked
protein ions in the gas phase [23-25]. Smith and coworkers have investigated the collision-induceddissociation processes of multiply-charged protein ions [26-29].
In the present work, the photo-induced reactions of multiply-charged cytochrome c ionsprepared by ESI are investigated with mass spectrometry. Cytochrome c is a relatively small protein
and has an iron-containing heme group covalently bound to two cysteines in a polypeptide chain of~100 residues. Since there are no disulfide bridges in cytochrome c, it is free to adopt conformations
that are quite different from the native conformations. We measure the photoionization yields at 28200
and 18800 cm-1 for the charge-selected cytochrome c ions, (M + nH)n+ (n = 9 – 17). Based on theseresults, we discuss the mechanisms of the photoionization and photodissociation of heme in relation to
the protein conformations.

9
13

13

8
10

7

11

6

12

5

4
N2

2
1

3

hν

Fig. 1. Schematic diagram of thetandem mass spectrometer with
ESI source;(1) electrosprayneedle movable via three axis
positioner, (2) interface plate, (3)heater, (4) aperture (0.1 mm i.d.),(5) skimmer and electrostatic
lenses, (6) first octapole ion-beam
guide(OPIG1),(7)
firstquadrupole
mass
filter(QMASS1), (8) first quadrupoleion deflector (QDF1), (9) second
octapole ion beam guide (OPIG2),(10) second quadrupole ion
deflector (QDF2), (11) second
quadrupole
mass
filter(QMASS2), (12) channeltron
with conversion dynode, (13)quartz window.

2. Experimental
Fig. 1 shows a schematic diagram of the apparatus consisting of an ESI source and a tandem
mass spectrometer with octapole ion beam guides. Multiply-charged protein ions are produced by ESIof a dilute solution of cytochrome c (Sigma-Aldrich) in methanol-water (1:1 v/v) mixture including
acetic acid (2.0%). Optimum intensity and stability of ion signals are obtained with a 1.2 × 10-4 M
solution of cytochrome c. The electrospray needle is biased at +7 ~ +9 kV with respect to a platinum
aperture of 0.1 mm in diameter located 15 – 20 mm from the needle tip. The platinum aperture isbiased +150 ~ +350 V against the ground. A flow of 10 – 100 mL/s of dry nitrogen is maintained
through the atmospheric pressure ion source. The flow continuously removes solvent vapor producedby the electrospray. Ions produced by the electrospray in the atmospheric region drift under the
influence of the electric field towards the interface plate, and enter the interface region through a hole
(4 mm in diameter) on the interface plate. The interface plate is heated up to about 350 K. Ions formed

at atmospheric pressure in the ESI source are admitted into the first vacuum chamber, which isevacuated by a 3000 L/s diffusion pump (ULVAC, ULK-10A). Typical pressure in the first chamber is
2.0 × 10-4 Torr. A beam of the ions then passes through a stainless-steel skimmer, and is focused byelectrostatic lenses into the first-octapole ion beam guide (OPIG1) [30]. The OPIG is composed ofeight molybdenum rods with 4 mm in diameter, which are evenly positioned on a circle of 16 mm indiameter. High transmission efficiency for slow ions is accomplished by applying a radio frequency(RF) field to the rods. The OPIG1 is connected directly to the first quadrupole mass spectrometer(QMASS1) (ABB EXTEL, 664901) in the second chamber evacuated by a 2000 L/s diffusion pump
(Edwards, Diffstak 250/2000C). Typical pressure in the second chamber is 5.0 × 10-6 Torr. The first
quadrupole ion deflector (QDF1) deflects the mass-selected ion beam emerging from QMASS1. The
ions are admitted into the second-octapole ion beam guide (OPIG2) with a length of 88 cm, which isplaced in the third chamber evacuated with a 400 L/s turbo molecular pump (SEIKO SEIKI, STP-400).
Typical pressure in the third chamber is 2.0 × 10-7 Torr. After passing through OPIG2, the parent andthe product ions are deflected by the second quadrupole ion deflector (QDF2), and are admitted into
the second quadrupole mass spectrometer (QMASS2) (ABB EXTEL, 652601). The ions are massselected and detected by a channeltron electron multiplier equipped with an ion-conversion dynode.
Depletion yield of the parent cluster ions in OPIG2 is measured with irradiating a photolysis laser. The
laser beam is introduced into the guide collinearly and counterpropergatedly with the ion beams. We
use the second (18800 cm-1) or third (28200 cm-1) harmonic of a YAG laser as the photolysis laser.
Relative photodepletion cross section, σrel, is determined byσrel = ln (Ioff / Ion)/φ ,(1)

where φ is the photon fluence, Ion and Ioff are the signal intensities of ions with and without irradiating
the photolysis laser, respectively.

Fig. 2. Typical mass spectrum ofmultiply-charged cytochrome cions produced by ESI as afunction of mass/charge ratio,
m/z.

15+

Ion Intensity / a.u.

14+
16+
13+

12+

17+

11+

700

800

900

1000

1100

Mass Number (m/z)

10+
1200

9+
1300

3. Results and Discussion
3.1. Products of Photo-Induced Reactions.
Typical mass spectrum of multiply-charged cytochrome c ions, (M + nH)n+, produced by ESI
is presented in Fig. 2 as a function of mass/charge ratio, m/z. The observed mass spectrum consists ofmultiply-charged ions ranging from the n = 9 to 17 charge states. Intensity and mass distribution of theionic species produced by ESI depend strongly on the electric field strength of the high-pressure zone
in the first chamber [31]. High electric field increases the kinetic energy of ion collisions with thebackground gas. By optimization of the electric field, ions of specific charge states are produced
efficiently. With increasing the electric field, intensities of smaller charge states are enhanced. In thepresent study, we do not observe metastable decomposition of the ions between the initial mass
selection and the final detection.
The photo-induced reactions of the charge-state selected cytochrome c ions are investigated at
18800 and 28200 cm-1. All charge states examined exhibit relatively strong photodepletion in both
excitations. The absorption bands in visible and UV regions of cytochrome c in solution are ascribedto the ππ* transition of heme. The spectrum exhibits the absorption bands of moderate intensity invisible region called as Q bands (ε ~ 2 × 104) and an extremely strong absorption band at ~ 25000 cm1, which is called as Soret band (ε ~ 3 × 105) [32]. The photo-induced reactions of the ions are
examined by the excitation of these transitions in heme.
The product ions are considered to be generated through the following processes; photoexcitationof heme in the protein ions (eq. 2), subsequent photoionization (eq. 3), or photodissociation (eq. 4), as
follows,(M + nH)n+ + m hν →→[(M + nH)n+]*→[(M + nH)n+]*[(M + nH)n+]*(M + nH)(n+1)++ efragments of dissociation

(2)
(3)
(4)

where [(M + nH)n+]* represents cytochrome c ions in the excited states. In this scheme, thephotoionization (eq. 3) competes with the dissociation (eq. 4). In relation to the third process, anumber of fragments have been observed via the cleavage of polypeptide chains in the collisioninduced dissociation (CID) of protein ions [19,26,29]. We would also expect to observe the similarfragmentation process for the photolysis of protein ions. In contradiction with this expectation, thefragment ions are not detected with sufficient intensity except for the aforementioned multiplycharged ions, (M + nH)(n+1)+. However, the results on the cross section of the photodepletion indicate
the contribution of third process as mentioned later.
In order to analyze the photodissociation process, we examine the branching fraction for theionization, Rion, as
Rion= Ipro[(M + nH)(n+1)+]/Idep[(M + nH)n+](5),

where Ipro[(M + nH)(n+1)+] is the ion intensity of the product ion, (M + nH)(n+1)+, whereas Idep[(M +nH)n+] is the depletion ratio of the parent ion. Fig. 3 shows the branching fractions of thephotoionization process plotted as a function of charge, n. Open squares and filled circles indicate thebranching fractions determined at 18800 (47 mJ cm-2 pulse-1) and 28200 cm-1 (7.8 mJ cm-2 pulse-1),
respectively. As shown in the figure, the branching fractions in both excitations decrease
monotonously with increasing the charge up to n = 16. The branching fractions are not dependent on
the laser fluence in both excitations. Smith and coworkers have reported the CID experiments ofmultiply-charged cytochrome c ions [26-28]. They have observed both charge transfer (CT, leading toloss of charge) and charge-stripping (CS, increase of charge by electron ejection) in addition to the
CID process to produce singly charged fragments. CID has been found to be dominant for the +14 to+19 ions, while the CT and CS processes are dominant for n = 9 – 12. The present
0.30

1.0

0.25
Cross Section/a.u.

Branching Fraction

0.8
0.6
0.4
0.2

0.20
0.15
0.10
0.05
0.00

0.0
9

10 11 12 13 14 15 16 17
Number of Charge (n)Fig. 3. Branching fractions for the photoionization ofmultiply-charged cytochrome c ions as a function of
charge. Open squares and filled circles show the
branching fractions determined with photolysis of thesecond (18800 cm-1) or the third harmonic (28200
cm-1) of YAG lasers, respectively.

9

10 11 12 13 14 15 16 17
Number of Charge (n)Fig. 4. Cross sections for the photodepletion ofmultiply-charged cytochrome c ions as a function of
charge. Open squares and filled circles indicate therelative cross sections determined with photolysis of
the second (18800 cm-1) or the third harmonic (28200
cm-1) of YAG lasers, respectively.

results are similar to those of the CID experiments. In Fig. 4, the relative cross sections of thephotodepletion of multiply-charged cytochrome c ions, (M + nH)n+, ( n = 9 – 17) are plotted as afunction of the charge. Open squares and filled circles indicate the cross sections determined at 18800(47 mJ cm-2 pulse-1) and 28200 cm-1 (7.8 mJ cm-2 pulse-1), respectively. As shown in the figure, thecross sections decrease gradually with increasing charges from n = 9 to 12, while they increase
gradually for n = 15 – 17 in both excitations.
Fig. 5 shows the laser fluence dependence on the photodepletion yield of cytochrome c ions(n = 14) examined at 28200 cm-1. The depletion yield increases in proportion to the 2.2 power of thelaser fluence; the reactions proceed via two-photon excitation. The photodepletion yield at 18800 cm-1
also increases in proportion to the 2~3 power of the laser fluence. In the present experiment, the pulse
width of the laser is 5 × 10-9 s, and the photon fluxes of the photolysis laser are 2.8 × 1024 cm-2 s-1 andCross Section / a. u.

2.6 × 1025 cm-2 s-1 at 28200 and 18800 cm-1, respectively. The ionization cross sections from the ππ*
excited states are assumed to be the same as that for the transition from the ground state; they are 1.1 ×
10-13 cm2 and 7.6 × 10-15 cm2 at 28200 cm-1 and 18800 cm-1, respectively. By using these values, the rate
constants for the ionization from the excited states are estimated as 3.0 × 1011 s-1 and 2.0 × 1011 s-1 at
28200 cm-1 and 18800 cm-1, respectively. Lifetime of the ππ* excited state of iron porphyrins insolution has been reported as 25-50 ps [33]. The rate constants of the ionization are nearly the sameorder as that of the decay process. Therefore, the observed photoionization of cytochrome c ions is
ascribed to the multiphoton excitation of heme.

4

Fig.

2

photodepletion of multiply-charged cytochrome cslope 2.2

0.1

5.

Laser

fluencedependence

for

the

ions (n = 14) at 28200 cm-1. The depletion yield
increases in proportional to the 2.2 power of the laser

6
4

fluence.

2
2

3
4
5
6 7 8 9 10Laser Fluence (mJ cm -2 pulse-1)

3.2. Model calculation of photoionization
As mentioned in the previous section, the photoionization (eq. 3) at 28200 cm-1 proceeds via
two-photon excitation of heme for n < 16. In order to clarify the origin of the size-dependence of thephotoionization yield, we carry out the model calculations. Since the polypeptide in cytochrome c ionshas positive charges, the photo-ejection of an electron (ionization) from heme takes place under the
influence of Coulombic attractive potential. In a crude approximation, the upper limit of the excessenergy, Eex, in the photoionization can be defied as
Eex = 2hν- IPheme(6),

where hν and IPheme are the photon energy and the ionization potential of heme in cytochrome cions. The photoionization may occur if the attractive potential becomes less than Eex. It has beenreported that heme iron in cytochrome c ions produced with ESI is the Fe (II) form [34,35], and thus,
heme in multiply-charged cytochrome c ions is electrically neutral. Ionization potential (IP) of the gasphase iron-porphyrins has been determined to be 6.06 eV (Fe (II) OEP) and 6.09 eV (Fe (III) ClTPP)
by the photoelectron spectroscopy [36] and photocurrent measurement [37], respectively. IP of hemein the ions may be roughly close to these values. Thus, Eex in the two-photon ionization at 28200 cm-1
(6.99 eV) is estimated to be ~ 0.9 eV. In the following sections, we calculate the attractive potentials
with structural models and compare the results with the above Eex.
3.2.1. Sphere ModelIn the first scheme, the Coulomb attractive potential is estimated by assuming that the shapes

of the protein ions are spheres. It is also assumed that the charges are distributed uniformly over thesurface of the sphere. Based on these assumptions, the Coulomb potential energy on the surface of thesphere, Usp, is expressed by

ne 2
Usp = −
6π ε 0 ε r rn(7),where n is the number of charges, εr is the dielectric constant of protein to account for the shielding of
the charges by protein itself, and rn is the radius of the sphere for the protein ion with the number ofcharge n. As for the relative dielectric constant, Williams and coworkers have suggested the value of ~2.0, which is appropriate for the gas-phase cytochrome c ion [22]. X-Ray crystal structure coordinatesand NMR solution structure coordinates, obtained from the Protein Data Bank, give a value for aneffective radius of the sphere, rn, as 9.25 Å for the native conformations [38]. If the conformation of
the gas-phase ions is the same as the native one, Usp is calculated to be 8.3 eV for n = 16, which ismuch larger than Eex (~ 0.9 eV). This result suggests that the Coulomb potential energy isoverestimated with this model. Jarrold and coworkers have examined the cross sections (Sn) of
cytochrome c ions in the gas phase as a function of charge by ion mobility measurements [23-25]. The
effective radius, rn = (Sn/4π)1/2, for the gas-phase ions is obtained as 14.7 Å. Using this value, the Usp
value is calculated to be 5.2 eV for n = 16, which is still larger than Eex. Therefore, the argumentssuggest that the conformation is quite different from sphere.

Heme

r

rb

x - axis
+

+

+

+

Polypeptide Chain

+

+

+

+

+

+

+

+

Fig. 6. Schematic of the scheme
to calculate Coulomb potential atheme, by assuming that thepolypeptide chain in the protein
ions is a linear string, and that the
charges are distributed uniformly
over the polypeptide chain.

x
dx

15ra

89ra

3.2.2. Linear string Model
The Coulomb attractive potential depends upon the conformation of polypeptide chain.
Cytochrome c has no disulfide bridges and is relatively free to adopt conformations, which are quite
different from the native conformation. In relation to this issue, the molecular dynamics simulations
for cytochrome c ions have predicted that the geometries close to the native structure in solution are
dominant for the low charge states (n = 3 - 5), while extended conformations are dominant for thehigher charge states (n = 7 - 20) [23-25]. These results suggest that cytochrome c ions in the present
experiments (n = 9 – 17) are completely unfolded. Cytochrome c is composed of 104 residues. Heme

is covalently bound to cysteine 14 and cysteine 17. In native conformation, heme iron is bound to thenitrogen and sulfur atoms in histidine 18 and in methionine 80, respectively [38]. When cytochrome c
unfolds, these ligands would dissociate from the iron. Thus, in the second model, the Coulomb
attractive potential is estimated by assuming that the polypeptide chain in the protein ions is a linear
string, and that the charges are distributed uniformly over the polypeptide chain along the x-axis asshown in Fig. 6. Contribution of these charges to the Coulomb potential per a unit length, dUstring, isestimated by the equation,dU string =−ρ e dx4 πε0εr r(8)where ρ is the charge density distributed over the residues in the polypeptide chain of the same
average size. ρ is given by

ρ=

ne
104ra

(9)

where n is the number of the charges, ra is the length of polypeptide chain per one residue, which isequal to 3.6 Å [39,40]. The distance between heme and the charges, r, is given byr = x 2 + rb 2

(10)

where rb is the distance between heme and the polypeptide chain. Because heme is bound to thepolypeptide chain with cysteine 14 and cysteine 17, the rb value is equal to 9.27 Å. By the integrationof dUstring along the x–axis, the Coulomb potential energy at heme, Ustring, is expressed as

Ustring = ∫

89 ra

0

dUstring + ∫

15 ra

0

dUstring .

(11)

The calculated Ustring values are plotted in Fig. 7 as a function of n. The Ustring value for n = 16 is given
as 0.89eV and is almost equal to the excess energy, Eex. For n < 16, the Ustring values become lower
than Eex and the ionization is expected to occur. These results are consistent with the present
experimental findings; the critical number of charge, n, for the ionization is 16. Therefore, thecalculations indicate that the shapes of the protein ions are similar to the linear string. Although thepresent calculations use the oversimplified models, the results are consistent with the observed
behaviors for the photoionization of cytochrome c ion.

Fig. 7. Coulomb attractive potentials at heme, which
are contributed by the charges on the linear
polypeptide chain, are plotted with filled circles. The
broken line indicates the excess energy, Eex, of twophoton ionization at 28200 cm-1.

Coulomb Potential (eV)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
10

12

14

16

18

20

Number of Charge (n)3.2.3. Cleavage of polypeptide chain
As mentioned in the previous section, the cross sections increase gradually for n = 15 – 17 inboth excitations, though the photoionization yields decrease monotonously in this size range. Although
no dissociation products are observed in the present experiment, these results clearly suggest theoccurrence of the dissociation of polypeptide chain as in the case of CID experiments [26, 27]. In
order to confirm the presence of the dissociation channel, we examine the repulsive interaction in themultiply-charged polypeptide chain, which may enhance the dissociation process. Relating to thisissue, Rockwood and coworkers have estimated the activation energy for the dissociation of gas-phase
protein ions by assuming a “charges on a string” model [41]. Using their model, we also estimate anenergy release upon dissociation. In the present calculations, the polypeptide chain in the protein ionsis assumed as a linear string, in which the charges are distributed uniformly over the residues in the
chain. The Coulomb repulsive potential between the two fragment ions, Urel, is estimated byUrel = −r1
r2 dx dxρ212∫∫4πε 0ε r 0 0 x1 x2(12)where r1 and r2 are the length of peptide chains for two fragment ions. The maximum value for the
repulsion potential, Urel(max), is obtained when the cleavage occurs at the bond in the middle of peptide
chain; r1 is equal to r2. For n = 17, the Urel(max) value is estimated to be 3.83 eV and suggest theenhancement of the bond dissociation. The gradual increase of the photodepletion cross section withincreasing n from 15 to 17 as seen in Fig. 4 may be ascribed to the increase of the dissociation yield asa result of the Coulomb repulsion. At the moment, we have no clear explanation for the absence of thefragmentation products. One of the possible reasons may be the broadening of the kinetic-energy
distribution of fragment ions in OPIG2, which may cause as a result of the energy release by Coulomb
repulsion upon dissociation and induces the reduction of the transmission efficiency of QDF2.

4. Conclusions
We have constructed the photodissociation mass spectrometer equipped with electrosprayionization source. Using this apparatus, photo-induced reactions of multiply-charged cytochrome cions, n = 9 - 17, have been investigated at 28200 and 18800 cm-1. The mass spectra of thephotoproducts show that the ionization occurs dominantly for the lower charged states. The laser
fluence experiments indicate that the ionization at 28200 cm-1 is two-photon process, while more
photons are required for the excitation at 18800 cm-1. A drastic decrease for the ionization yields isobserved with increasing the charge states, n. This change is interpreted by the ionization of heme
under the influence of the Coulomb attractive potential in the polypeptide chain. Model calculations
for the Coulomb potential suggest that structure of the polypeptide chain is completely elongated.
Acknowledgements
This work is partially supported by the Grant-in-Aid (Grants #11304042) from the Ministryof Education, Science, Sports and Culture of Japan, and by the Grant-in-Aid for Scientific Research(Grants #11740326, and #13640512) and a research grant for the future program from Japan Society
for Promotion of Science. KF is also grateful to the Hyogo Science and Technology Association forpartial financial supports.

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