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Electron Transfer in Bacterial Reaction Centers with the Photoactive Bacteriopheophytin Replaced by a Bacteriochlorophyll through Coordinating Ligand Substitution Jie Pan, Rafael Saer, Su Lin, J. Thomas Beatty, and Neal W. Woodbury Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00317 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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Biochemistry

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Electron Transfer in Bacterial Reaction Centers with the

2

Photoactive Bacteriopheophytin Replaced by a

3

Bacteriochlorophyll through Coordinating Ligand

4

Substitution

5 Jie Pan 1 #, Rafael Saer 3 ¶, Su Lin 1, 2, J. Thomas Beatty 3, Neal W. Woodbury 1, 2*

6 7 8

1

9

85287-5201;

The Biodesign Institute at Arizona State University, Arizona State University, Tempe, Arizona 2

School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287-

10

1604; 3 Department of Microbiology and Immunology, The University of British Columbia,

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2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3 #

12 13 14

¶

Current address: Physics department, Florida International University, Miami, FL 33199

Current address: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130

15

1

ACS Paragon Plus Environment

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KEYWORDS

2

Bacterial reaction center; β-type mutant; protein dynamics; electron transfer kinetics and

3

pathways; protein relaxation; charge recombination.

4 5

ABBREVIATIONS

6

BChl, bacteriochlorophyll; BPhe, bacteriopheophytin; RC, reaction center; P, primary electron

7

donor; BA and BB, monomer bacteriochlorophylls; HA and HB, bacteriopheophytins; QA and QB,

8

quinones; DAS, decay associated spectra; ps, picosecond; ns, nanosecond.

9 10

2


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Abstract

2

The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate

3

and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial

4

photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides

5

mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H),

6

glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN,

7

respectively. As has been reported previously for M214LH, each of these mutations resulted in a

8

bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-

9

called β-type mutants (in which the HA cofactor is called βA). In addition, these mutations varied

10

the properties of the surrounding protein environment in terms of charge distribution and the

11

amino acid side chain volume. Electron transfer reactions from the excited primary donor P to

12

the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques.

13

Similar to the previously characterized M214LH (β-mutant), the strong energetic mixing of the

14

P+BA– and P+βA– states (the mixed anion is denoted I–) increased the rate of charge

15

recombination between P+ and I– in competition with the I– → QA forward reaction. This reduced

16

the overall yield of charge separation forming the P+QA– state. While the kinetics of the primary

17

electron transfer forming P+I– were essentially identical in all three β-mutants, the rate of the βA–

18

(I–) → QA electron transfer in M214LQ and M214LH were very similar but quite different from

19

that of the M214LN mutant. The observed yield changes and the differences in kinetics are

20

correlated more closely with the volume of the mutated amino acid than with their charge

21

characteristics. These results are consistent with previous studies of a series of M214 mutants

22

with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan 3


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et al., 2013 JPCB]. Both studies indicate that protein relaxation in this region of the reaction

2

center plays a key role in stabilizing charge separated states involving the HA or βA cofactor. The

3

effect is particularly pronounced for reactions occurring on the tens and hundreds of picosecond

4

timescales (forward transfer to the quinone and charge recombination).

5

4


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Biochemistry

Introduction

2

The multi-step design of photosynthetic electron transfer is a key feature in enabling near unity

3

quantum efficiency of charge separation in reaction centers (RCs). The probability of a wasteful

4

back reaction is minimized at each intermediate state by the protein environment, which controls

5

the energetics of the process both statically and dynamically. In the RC of Rhodobacter (Rb.)

6

sphaeroides, the electron transfer process is initiated following the excitation of a

7

bacteriochlorophyll (BChl) dimer, P, yielding P*. An electron is transferred from P* to a

8

bacteriopheophytin (BPhe), HA, via a monomeric BChl, BA, followed by electron transfer to a

9

quinone, QA, and finally to a second quinone, QB (Figure 1A) 1-4. The back reaction at each step

10

is 2 to 4 orders of magnitude slower than the forward reaction, resulting in a near-unity quantum

11

yield. The BPhe in the HA site participates in at least three reactions over multiple timescales: (i)

12

receiving an electron from BA– on the picosecond (ps) timescale, forming P+HA–; (ii) transferring

13

an electron to QA on the hundreds of ps timescale, forming P+QA–; and (iii) undergoing charge

14

recombination from HA– to P+ with a time constant on the nanosecond (ns) timescale 5-16.

15

In biological electron transfer systems, protein dynamics play an essential role in stabilizing

16

charge separated states. Reactions that occur on hugely different timescales likely experience

17

different types of protein motion, and this appears to play a critical role in RCs 17-22. In the wild

18

type Rb. sphaeroides RC, the 200-ps forward reaction from HA– to the first quinone QA out-

19

competes the 10 to 20 ns P+HA– recombination reaction almost completely. This is at least in

20

part because the relative free energy of P+HA– decreases with time due to a relaxation of the

21

protein environment over hundreds of picoseconds to nanoseconds, preventing charge

22

recombination via the P+BA–state 10, 12, 20, 23-27.

5


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The kinetics of RC electron transfer have been extensively studied as a function of driving

2

force by varying the relative energetics between the cofactors via genetic or chemical

3

modification

4

was observed in the so-called β mutant (M214LH), in which the BPhe in the HA site is replaced

5

by a BChl molecule (denoted as β), resulting in a nearly zero driving force between P+BA– and

6

P+βA– 33. Because these two states have very similar energies, they are often both populated and

7

the mixture sometimes referred to as P+I-. Kirmaier et al. observed that the overall forward

8

electron transfer rate from P* to βA decreased by approximately two-fold, and that the electron

9

transfer time constant to QA slowed from 200 ps (with HA–) to 580 ps (with βA–)

23, 28-32

. In particular, a significant impact on the HA– → QA electron transfer rate

28, 29

. Charge

10

recombination from the state P+βA– occurs with a time constant of about 1 ns, an order of

11

magnitude faster than that in the wild type RC, resulting in a 40% decrease in the yield of the βA–

12

→ QA reaction

13

the mutant M214LH+L104EV resulted in an even larger βA– → QA yield loss (70%)

14

Interestingly, RCs containing Zn-BChls in place of all six chlorins (four BChls and two BPhes)

15

retain essentially wild type electron transfer rates even though the A-branch cofactor

16

composition is similar to the above-mentioned β mutant RC

17

was that the coordination state of the metal in the Zn-BChl in the HA pocket was such that the

18

energy gap between the P+BA– and P+HA– states remained similar to wild type

19

explanation was further supported by recent work that compared the wild type RC and the

20

M214LH mutant (β-mutant) with the Zn-RC and a Zn-RC β-mutant 37.

21 22

28

. Further elevation of the energy level of βA by removing a hydrogen bond in

35

28, 34

.

. The interpretation of this result

35, 36

. This

The coupling between protein dynamics and electron transfer has also been shown to be essential in controlling the rate of formation and yield of the charge separated state P+QA–

25, 38,

6


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39

2

highlighted the role of protein dielectric relaxation on the timescale of QA– formation

3

Experimentally, the effects of altering protein dynamics on the HA– → QA electron transfer

4

kinetics and yield were explored in a study of a series of mutants at M214, altering the

5

immediate environment of HA 41. Amino acids with a series of different molecular volumes were

6

substituted at M214 site (methionine (M), glycine (G), alanine (A) and cysteine (C)), and

7

compared to the native leucine (L). Unlike the β-mutant, these mutants do not result in the

8

presence of BChl in the HA site. Instead, BPhe was present, and the effects on electron transfer

9

were interpreted in terms of altered protein dynamics in the region due to changes in the volume

10

. A recent theoretical treatment of the role of protein dynamics in RC electron transfer 40

.

of the amino acid side chain, which changed protein flexibility near the HA site.

11

Although P* → HA electron transfer occurs on a ps timescale where protein movement is very

12

limited, the HA– → QA electron transfer reaction occurs in hundreds of ps, a timescale in which

13

collective protein motions begin to come into play

14

potential for protein dynamics to affect the rate of forward electron transfer from HA– → QA, and

15

charge recombination of P+HA–, than P* → HA electron transfer. To further understand the

16

extent to which protein dynamics near HA determines the rates and pathways of electron transfer,

17

as opposed to other aspects of the protein environment, the kinetics of each step in the P* → QA

18

electron transfer reaction were examined in a set of M214 β-mutants with amino acid residues

19

that have altered electronic/chemical properties in addition to different side chain volumes.

12, 24, 40, 42, 43

. Thus there is a much greater

20 21

Materials and Methods

22

M214 mutant reaction centers. Procedures for preparing wild type and mutant RC proteins have

23

been described previously

41

. The plasmid pAli2, containing the pufQBALMX genes of Rb. 7


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sphaeroides on a 4.6-kb EcoRI fragment, was modified by site-directed mutagenesis using the

2

primer

3

complement, where XXX represents the codon for histidine (H), glutamine (Q), or asparagine

4

(N). The desired changes were confirmed by DNA sequencing of both the pufL and pufM genes.

5

The mutant pufQBALMX operons were subcloned into a derivative of plasmid pRS1 37, which

6

contains a modified (RC H-subunit) gene encoding a C-terminal 6-histidine tag. The puhA gene

7

is positioned transcriptionally upstream of the mutant pufQBLAMX operon. Expression of RC

8

constructs was driven by the presence of the hypoxia-inducible puc promoter upstream of the

9

puhA gene. Purification of RCs was carried out according to a modified version of a published

10

protocol 44. For spectroscopic measurements, RCs were in a solution of in 10 mM Tris-HCl (pH

11

8.0), 0.1% LDAO, and 10 mM orthophenanthroline to block the QA to QB electron transfer.

5’-CTCTACGGGTCGGCCXXXCTCTTCGCGATGCAC-3’,

and

its

reverse

12 13

Femtosecond Transient Absorption Spectroscopy.

The femtosecond transient absorbance

14

spectrophotometer has been described previously 45. Excitation pulses at 865 nm were generated

15

from an optical parametric amplifier (OPA-800, Spectra-Physics) pumped by a kilohertz

16

regenerative amplifier system (Tsunami and Spitfire, Spectra-Physics). Transient absorption

17

changes at various wavelengths were measured using a spectrophotometer coupled with a CCD

18

camera (DU420, Andor Technology). The polarization of the pump pulses was set to the magic

19

angle (54.7°) with respect to that of the probe pulses. Absorbance change spectra as a function of

20

time delay were recorded in both the QX (500~760 nm) and QY (680~980 nm) transition regions

21

of the RC bacteriochlorins. RC samples were loaded in a spinning wheel with an optical path

22

length of 1.2 mm at a final optical density of ~0.8 at 800 nm. All measurements were performed

8


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Biochemistry

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at room temperature. Time-resolved spectra were corrected for spectral dispersion, and data

2

analysis was performed using a global fitting program as described previously 46.

3 4

Results and Discussion

5

Ground State Absorption Spectroscopy

6

The absorption spectra of the M214LH, M214LQ, and M214LN mutant RCs are compared

7

with that of the wild type RC in Figure 1B (upper panel). All spectra are normalized to the P

8

band in the QY transition region at 865 nm. A linear background subtraction was performed for

9

the regions between 460 to 640 nm and 700 to 940 nm. In the wild type RC, the QY transitions of

10

the H, B and P cofactors have well-separated bands, peaking at 765, 802 and 865 nm,

11

respectively. The absorption bands around 540 and 600 nm are due to the QX transitions of H,

12

and B/P, respectively. Difference spectra between mutant and wild type RCs show decreased

13

absorption in the M214 substitutions (H, N and Q) near 545 and 760 nm (BPhe) and increased

14

absorption near 600 and 780 nm (BChl) (lower panel, Figure 1B). These changes are similar to

15

those previously observed for the β-mutant M214LH

16

interpretation that BChl is present in place of Bphe at the HA site in the M214LN and M214LQ

17

mutants. The difference spectra also show the relative QX and QY transition energies of the β

18

cofactor between the three mutant RCs. In the QX transition region (590 to 600 nm), the

19

M214LN mutant exhibits the highest transition energy (~590 nm), with the M214LQ (595 nm)

20

and M214LH (597 nm) mutants closer to the wild type transition energy (600 nm). In the QY

21

region, there appear to be two spectral components in the difference spectra between 770 and

22

800 nm where the β-cofactor is expected to absorb (Figure 1B, lower panel). Both transitions

23

have the highest energies in the M214LQ mutant (775 nm and 800 nm) and the lowest energies

28, 29

and are consistent with the

9


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in the M214LN mutant (787 nm and 804 nm), with the M214LH mutant intermediate but closer

2

to the M214LQ mutant (783 nm and 800 nm).

3 4

Primary Charge Separation in the β-type Mutants

5

Electron transfer dynamics in this series of β-mutants was evaluated by femtosecond transient

6

absorption spectroscopy of purified RCs. Absorbance changes were recorded between 500 and

7

980 nm, and from 0.5 ps before to 6 ns after photoexcitation of the primary electron donor, P, at

8

865 nm.

9

The primary charge separation event after photoexcitation of P was monitored as the decay of

10

stimulated emission from P* near 920 nm, and a significantly slower decay was observed in all

11

three of the M214 mutants compared to the wild type RC (Figure 2A). The decay traces of the

12

mutants are nearly identical for the first 25 ps (Figure 2A, inset, left panel). The P* kinetics in

13

all mutants and the wild type RC can be fitted to 2 lifetimes, one around 3 ps and the other

14

between 10 and 15 ps (Table I), but there is a substantial increase in the relative amplitude of the

15

longer (10 to 15 ps) component in the mutants, as reflected in the calculated average lifetimes

16

given in Table I. These results imply that all the β-mutants undergo primary charge separation

17

with essentially the same kinetics, which is slower than that of the wild type RC. The results

18

agree well with the previously reported two-fold decrease in the overall initial electron transfer

19

reaction rate in an M214LH mutant 28, 37. The kinetic traces for the three mutants at 920 nm show

20

significant differences beyond 25 ps (Figure 2A, inset, right panel), a feature which will be

21

discussed below.

22

Figure 2B shows a comparison of the transient absorption difference spectra of the wild type

23

and the three mutant RCs in the QX transition region at 25 ps after excitation. At this time, the 10


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Biochemistry

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spectra represent the early charge separated state, P+HA– or P+I– for the wild type or the M214

2

mutant RCs, respectively. Amplitudes of the mutant spectra were normalized to the absorbance

3

difference observed in the wild type at 500 fs and 600 nm (Figure 2B, inset), adjusting for the

4

initial P* population. These scaling factors were then applied to the entire corresponding data set.

5

The wild type spectrum at 25 ps is characterized by the ground state bleaching of P and HA (a

6

BPhe molecule) at 595 nm and 540 nm, respectively, and an absorption increase peaking at 665

7

nm due to the absorption of the HA– anion radical. In the β-mutants, the cofactors at the BA and

8

HA sites are BChl molecules, and thus the states P+BA– and P+βA– are energetically very close and

9

spectrally indistinguishable. The strongly mixed P+BA– and P+βA– states are therefore denoted as

10

P+I–. All three mutants show similar transient absorption spectra at 25 ps, but differ from that of

11

wild type RCs in several important respects. The 540-nm bleaching band due to HA– formation in

12

the wild type RC is missing, and the amplitude of the bleaching at 600 nm is almost doubled in

13

the mutants compared to wild type. In addition, the 600-nm absorbance decrease observed in all

14

samples was found to be shifted by 5 nm to the blue in the M214LN mutant, in accordance with

15

a similar shift observed in the ground state absorption spectrum of this RC (Figure 1B), which

16

likely originates from ground state bleaching of β at the HA site when the P+βA– state is formed.

17

The broad absorption increase in the 620 to 700 nm region exhibits two peaks in the mutants at

18

630 and 680 nm, which are the spectral features of the I– anion. The P+ cation band at 700 nm

19

overlaps with the I– signal, based on previous studies and in the wild type data shown in Figure

20

3A. These spectral features agree with the published results from M214LH mutants

21

yield of P+I– appears to be identical for the M214LH and M214LN mutants, but the observed

22

absorbance changes at 600 and 680 nm for the M214LQ mutant suggest a 5% yield loss.

28, 37

. The

23 11


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Electron Transfer on the Hundreds of ps-to-ns Timescale

2

Repopulation of P* on the hundreds of picoseconds timescale. In contrast to the similar

3

kinetics observed for the initial charge separation in all three β-mutants, the reactions occurring

4

on the hundreds of picoseconds to nanosecond timescale differ significantly both between the

5

mutant and the wild type RCs, and between the individual mutants (M214LH/M214LQ and

6

M214LN). This is evident in the complex kinetic behavior in the 920 nm region on the

7

nanosecond timescale, which can be seen clearly on an expanded intensity scale (Figure 2A,

8

inset, right panel). For the wild type RC the 920 nm signal is initially negative, becomes positive

9

after about 20 ps, and then becomes negative within a few hundred ps. We attribute these

10

changes to the increase and loss of absorbance from the HA– anion as the electron goes from P*

11

→ HA → QA. The signal remains positive for several nanoseconds in the quinone-depleted wild

12

type RC because HA– lives longer in the absence of a quinone acceptor. In contrast, the 920 nm

13

signal in M214LH and M214LQ does not become positive during the first 1 ns, while in

14

M214LN, the signal becomes positive and stays positive out to 2 ns. All the β-mutant RCs have

15

BChl in place of BPhe in the HA site and thus do not form HA–; therefore, there must be

16

something other than the cofactor substitution that causes the difference between the M214LN

17

and the other 2 β-mutants.

18

A slow decay phase observed in the time-resolved fluorescence at 920 nm, with a lifetime of

19

0.8 to 1 ns, was previously reported in the M214LH β-mutant and was attributed to delayed

20

repopulation of P* (giving rise to stimulated emission) resulting from thermal repopulation of P*

21

from P+I– charge recombination 28. The kinetics at 920 nm for M214LH and M214LQ shown in

22

Figure 2A are consistent with the previous observations of the M214LH RC, and indicate that a

23

significant amount of P+I– recombination takes place, resulting in a negative signal at 920 nm 12


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Biochemistry

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due to the stimulated emission of P*. The long-lived bleaching on the hundreds of ps timescale is

2

not observed in the M214LN mutant RC. Instead, the initial bleaching of the M214LN RC at 920

3

nm recovers and becomes positive as observed in the wild type RC, but remains positive after 50

4

ps and decays slowly, on the timescale of P+I– recombination. As described below, this is likely

5

due to the energetics of P+I– in the M214LN mutant RCs.

6 7

P+QA– yield. In the original β-mutant, M214LH, the cofactor composition change from a BPhe

8

to a BChl molecule at the HA site results in a decreased rate of forward electron transfer from βA–

9

to QA and an acceleration of P+βA– recombination, likely via P+BA–

28, 47-49

. This leads to a

10

decrease in the overall quantum yield of P+QA– formation. The M214LQ and M214LN mutants

11

also appear to have decreased yields of P+QA– formation. This can be seen in Figure 3A by

12

comparing the transient absorption spectra of the wild type and the three M214 mutant RCs

13

recorded at a 4-ns delay. In the wild type RC, electron transfer from HA– to QA occurs in 200 ps.

14

Thus the HA ground-state bleaching at 540 nm and the anion absorbance increase at 665 nm have

15

completely disappeared by 4 ns, leaving predominantly the spectral features due to P+ with an

16

absorption decrease at 600 nm and a small increase around 700 nm (Figure 3A, black curve). A

17

significant decrease in the amplitude of these spectral features is observed at 4 ns in the M214LH

18

and M214LQ mutant RCs, indicating the loss of I– and P+ (Figure 3A, dark cyan and blue

19

curves). The P+ loss alone can be seen clearly in the QY spectral region (865 nm bleaching) in

20

Figure 3B. For the M214LN mutant, the amplitude of the 600 nm bleaching band is much more

21

pronounced than it is in M214LH and M214LQ, and the shape of the difference spectrum for

22

M214LN at 4 ns, is very similar to the 25 ps spectrum in this mutant, though smaller in

23

amplitude (compare Figure 3A and Figure 2B). The x-ray structure of the M214LN mutant 13


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shows a ubiquinone molecule in the QA site

, but apparently electron transfer to QA is

2

incomplete in this mutant and a significant amount of P+I– remains on the ns timescale. The

3

difference in this regard between the M214LN mutant and either the M214LQ or the M214LH

4

mutant can be seen more clearly when the 4 ns spectra are normalized to the P+ bleaching

5

maximum near 600 nm in the three mutants (Figure 3A, inset); a very low level of I– is present in

6

both M214LH and M214LQ, judging from the greatly decreased anion band in the 620 to 680

7

nm region. In addition, the bleaching near 600-nm is blue-shifted more than 5 nm in M214LN,

8

compared with the bleaching in WT and the other 2 mutant RCs. This spectral signature is

9

consistent with that of the BA anion band observed in the WT RC and BChl in solution

51

,

10

together with the absorption increases in the 620 – 700 nm region, suggesting that a significant

11

amount of the mixed state P+I– exists and that it is likely dominated by P+β– .

12 13

State(s) formed at nanosecond delay times. The differences in kinetic behavior of the RCs

14

studied here can also be seen in the decay of the HA– anion signal (I– for mutant RCs) on the ns

15

timescale in Figure 3C, where the kinetic traces have been normalized at their maxima. In the

16

wild type RC (black curve), the HA anion signal decays mono-exponentially with a time constant

17

of 200 ps due to the electron transfer from HA– to QA. The M214LH and M214LQ mutants show

18

both a slower rise and a slower decay of the I– signal, with a decay time constant of 500 to 600

19

ps. A more complicated decay is observed in the M214LN mutant (Figure 3C, red curve) where

20

about 40% of the I– signal decays on a hundreds of ps timescale, while another 30% decays on

21

the ns timescale, leaving roughly 30% of the signal remaining at 6 ns. The fact that the P+I– is so

22

long-lived in the M214LN mutant implies that the electron transfer and recombination kinetics

23

are quite different from that of the wild type, M214LH and M214LQ mutant RCs. 14


Page 15 of 36

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Biochemistry

1

P+ yield loss was also observed by monitoring the ground state bleaching of P in the QY

2

spectral region (865 nm) at 4 ns (Figure 3B). As with the QX transition region, each data set was

3

scaled so that the initial population of P* at 0.5 ps was the same in all samples. The kinetics of

4

ground-state bleaching of P, represented by the normalized traces at 830 nm of all three M214 β-

5

mutants are compared to that of the wild type RC in Figure 3D. In the wild type, an essentially

6

constant bleaching (only a slight amplitude increase after 100 ps) persists over the entire 6-ns

7

time window, consistent with a 100% yield of electron transfer from HA– to QA, forming the

8

P+QA– state that is stable for 100 ms 20. The P-band bleaching shows substantial recovery on the

9

nanosecond timescale in both the M214LH and M214LQ mutants, with a rate constant similar to

10

the recovery of the I– anion signal in Figure 3C. The amount of P ground state bleaching at 4 ns

11

for M214LH, however, is only half of that in M214LQ (Figure 3B). The P+ bleaching in the

12

M214LN RC also recovers, but has done so to a lesser extent than the other mutants at 4 ns,

13

implying that a substantial P+ component remains on this timescale. The percentage of P+

14

remaining at 4 ns in the three mutants, estimated from the normalized P-band bleaching at 865

15

nm (P+ signal, Figure S1) is 17%, 35%, and 62%, for M214LH, M214LQ, and M214LN,

16

respectively. The 4-ns spectra of M214LH and M214LQ show mainly the signatures of P+QA–,

17

similar to the non-decaying spectrum of WT RCs. In contrast, the 4-ns spectrum of the M214LN

18

prominently displays the spectral features of P+I– with positive signals in the 630 – 680 nm

19

region and a negative band around 790 nm, both from I–.

20 21

Global Analysis and Possible Reaction Mechanisms in the β-mutants

22

To associate the observed spectral and kinetic changes induced by mutations with possible

23

changes in the electron transfer reactions, global analysis using multiple exponential decay 15


Biochemistry

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Page 16 of 36

1

components was performed over the entire QX and QY transition regions from 520 to 970 nm for

2

all RC samples. The resulting time constants and the corresponding decay associated spectra

3

(DAS) are given in Figure 4. For all three β-mutant RCs, 5 components were necessary to fit the

4

data in both the QX and QY regions. Although four components were adequate to fit the wild type

5

dataset, five exponentials were used for a parallel comparison to the mutants. The time constants

6

obtained from all samples were in the ranges of 3 ps (black), 10 to 15 ps (red), 177 to 228 ps

7

(green), 0.8 to 2.6 ns (orange), and 10 ns to 1 µs (blue). The two short-lived components (A to

8

D) and the three longer-lived components (E to H) from each sample are shown separately for

9

clarity.

10 11

Formation of the P+I– state. The decay-associated spectrum of the 2.7-ps component in the wild

12

type RC (black curve, Figure 4A) shows positive bands at 540, 600, 760 and 810 nm, and a

13

negative band at 660 nm, associated with the formation of P+HA–. The negative band at 900 nm

14

represents the disappearance of P* (stimulated emission). The 3-ps DAS traces (black) from the

15

three β-mutants have similar spectral features, showing positive bands around 600 and 810 nm

16

and a broad negative band in the 620 to 700 nm region, and a shoulder on the shorter wavelength

17

side of the 810 nm band. However the 3-ps DAS of the wild type shows a bleaching at 540 nm

18

which is not seen in the mutants. Additionally, the positive band at 760 nm has a lower

19

amplitude and is shifted to 740 nm, and there is a bleaching at 770 nm in the mutants not present

20

in the wild type. Those 3-ps features are due to the formation of P+I– and the replacement of the

21

BPhe electron acceptor in the wild type RC with a BChl molecule in the β-mutants. The decay

22

kinetics of the stimulated emission from P* peaking at 900 nm in the three mutants are similar to

23

that of the wild type RC. The major features of the 10- to 15-ps DAS (red) of each sample is 16


Page 17 of 36

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Biochemistry

1

spectrally similar to the 3-ps DAS, indicating that this feature is simply another kinetic

2

component of the same process (P* → P+HA–/ P+I–), as observed in previous studies

3

The amplitude of the 10- to 15-ps component relative to the 3-ps component increases in the

4

order: wild type; M214LH; M214LQ; M214LN; resulting in an overall decrease in the average

5

rate of the P* → P+I– reaction in all three β-mutants, as described above and in Table I and

6

Figure 2A. The 600-nm band in the 12-ps DAS of the M214LN mutant is blue-shifted from its

7

position in the 3-ps DAS. This taken together with the fact that the QX band of the β cofactor in

8

the M214LN mutant absorbs on the blue side of the 600-nm band at low temperature in the

9

ground state absorbance spectrum

41

25, 37, 52, 53

.

suggests that the equilibrium between P+BA– and P+βA– is

10

shifted toward P+βA– on the 10-ps timescale in this mutant. Another distinguished spectral

11

feature between the WT and mutant RCs is in the QY region. For WT RCs, the 10-ps DAS

12

maintains a spectral profile similar to that of the 3-ps DAS, while the 12-15 ps DAS of all 3

13

mutants exhibit a major feature at 760 nm which distinguish the 2 DAS. The absorption increases

14

between 700 – 810 nm is likely due to the formation of I–.

15

The M214LH mutant has been studied previously alone, as well as with additional mutations

16

28, 37

17

for each reaction pathway. Based on the reaction scheme adopted from Kirmaier et al. for the

18

M214LH mutant 28, our average lifetimes given in Table I for initial electron transfer agree well

19

with the published result for M214LH. In general, replacing the L residue at M214 with H, Q, or

20

N results in a two-fold reduction of the average rate of the first electron transfer step, but the

21

yield is maintained at almost 100%.

. Comprehensive kinetic models have been utilized to obtain microscopic kinetic constants

22

17


Biochemistry

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Page 18 of 36

1

P+ yield loss and the state(s) remaining on the ns scale. Much larger differences between the

2

wild type and mutant RCs, as well as among the three mutants, were observed in the hundreds of

3

ps and the ns DAS (Figure 4E to 4H) than at the shorter timescales. In the wild type RC, the 220

4

ps (green) DAS reflects the spectral changes associated with the P+HA– → P+QA– reaction, and

5

the non-decaying (blue) DAS is representative of the P+QA– state that lives for 100 ms in the

6

absence of QB

7

type RC indicates that there is no significant change in the amount of P+ during the HA– → QA

8

electron transfer. The positive band peaking at 910 nm is due to the loss of HA–, as shown in the

9

920-nm kinetics in the inset of Figure 2B and observed previously

20

. The small positive amplitude around 865 nm in the 220-ps DAS of the wild

51

. For comparison to other

10

samples, the wild type data was fit with 5-exponentials resulting in a 1 ns component not present

11

when fewer fitting terms are used. However this component does not statistically improve the fit

12

and has a near zero amplitude across the entire wavelength region.

13

It contrast to wild type, the amplitude of the DAS traces with near 1 ns lifetimes for M214LH

14

and M214LQ RCs are substantial (the lifetimes are 0.8 and 0.9 ns, respectively; shown as orange

15

lines in Figure 4F and 4H). Fits of the M214LH and M214LQ mutants also result in DAS with

16

lifetimes of 180/230-ps. Both the 180/230-ps and the 800/900-ps DAS show similar spectral

17

profiles and time constants, though the mutants differ in relative amplitudes of these

18

components. The longest decay DAS of both mutants has spectral features indicative of P+QA–.

19

The yield of this state is about 25% (for M214LH) and 40% (M214LQ), calculated using

20

equation =

21

values agree well with the amplitude decreases of the P-band from 25 ps to 4 ns, observed in the

22

time-resolved spectra (Figure 3 and Figure S1). To further explore the intermediate states

23

contained in the 200-ps and 1-ns DAS, the long-lived DAS in Figure 4E to H for each sample

, where Ai is the amplitude of P-band at 870 nm in the ith DAS. The

18


Page 19 of 36

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Biochemistry

1

have been normalized to the P-band bleaching at 870 nm to compare the changes in the spectral

2

features (Figure S2). The 180- and 220-ps DAS of the M214LH and M214LQ mutants (Figure

3

4B, green curves) show not only the yield loss of P+ evidenced by the amplitude of the P-band

4

bleaching at 600 and 870 nm, but also indicate a substantial decrease in the I– signal between 620

5

to 700 nm (decay of the anion absorbance). The spectral features between 620 to 700 nm can be

6

seen more clearly in the expanded scale shown in Figure S2. In addition, the profile of the ~200

7

ps DAS between 740 and 820 nm are nearly opposite of those seen in the longest-lived DAS

8

(P+QA–). These spectral features are consistent with the ~200 ps DAS representing both some

9

forward electron transfer from P+I– → P+QA and some P+I– recombination. The 800- to 900-ps

10

DAS contains most of the spectral features of the 200-ps DAS, except for the higher amplitude at

11

640 nm and a red shift of the negative band around 800 nm. In addition, the P band and the I–

12

anion band in the 800- to 900-ps DAS are of much greater amplitude in the M214LH mutant

13

than in the M214LQ mutant (Figure 3A and 3B), indicating that the P+I– recombination on the

14

800 to 900 ps timescale dominates for M214LH. The P+QA– yield in the M214LH is about half of

15

that reported by Kirmaier et al 28. The loss of P+I– via the recombination of P+ and I– resulted in

16

a thermal repopulation of P* and a reduced P+ QA– yield which is reflected in the kinetics at 920

17

nm shown in the right inset in Figure 2A.

18

The hundreds of picoseconds and the nanosecond DAS for the M214LN RC are different from

19

that of all other RCs in this study (Figure 2G). The 220-ps component (green curve) shows

20

signals associated with the P+I– → P+QA electron transfer reaction in the 620 to 700 nm region,

21

but the amplitudes of the ground state recovery of P at 870 nm are smaller than in the M214LH

22

or M214LQ mutants, indicating a higher amount of P+ remaining within the first 200 ps. The

23

signal losses at 600 and 800 nm, as well as from 620 to 720 nm are suggestive of P+I– → P+QA 19


Biochemistry

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Page 20 of 36

1

electron transfer. Comparing the 2.6-ns DAS to that of the 200-ps DAS, the differences in the

2

ratio of P-band bleaching at 870 nm to the positive signals in the 620 to 700 nm region suggest

3

that more P+I– recombination occurs on the nanosecond timescale. Note that the lifetime of the

4

second DAS (orange curve) is more than two times greater than those of the M214LH and

5

M214LQ mutants (2.6 ns vs. ~1 ns). In the M214LN mutant, about 60% of the P+ state persists in

6

the non-decaying DAS, judging from the amplitude of P+ at 870 nm. In contrast with the longest-

7

lived DAS in the other two β-mutants, substantial I– signal was observed in M214LN (positive

8

signal in the 620 to 720 nm region). To estimate the relative amount of P+I– and P+QA– states in

9

the non-decaying DAS of this mutant, the ratios of the bleaching amplitude at 600 and 870 nm

10

were determined. For the M214LH and M214LQ mutants, the ratios are 0.125 and 0.118,

11

respectively. Assuming that these mutants are entirely in the state P+QA– after several ns and thus

12

a ratio of 0.12 is indicative of the relative amount of QX and QY P-band bleaching in the P+QA–

13

state, the ratio of 0.22 found from the non-decaying DAS of M214LN indicates that of the P+

14

remaining in the non-decaying DAS, 45% is P+I–and the remaining 55% is P+QA–. The final yield

15

of P+QA– along the P+I– → P+QA– reaction pathway is marked in Figure 5 for each RC sample.

16 17

Reactions from P+I–. In previous studies of the M214LH mutant, an increased lifetime of P+I–

18

was observed, from 200 to 350 ps, resulting in a 30% yield loss of P+QA– formation due to

19

competition with P+I– recombination 28. The results for the 3 β-mutants in the current study also

20

show an overall increase of the average P+I– lifetime, on the order of 500 to 600 ps. This long-

21

lived P+I– component is heterogeneous, can be further resolved into exponential lifetimes of 200

22

ps and a longer lived component in the range of 0.8 to 2.6 ns. Simplified kinetic models, adapted

23

from the work by Kirmaier et al.

11

, were used to compare the differences between mutants 20


Page 21 of 36

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Biochemistry

1

(Figure 5). While these schemes do not quantitatively represent the total heterogeneity of the

2

electron transfer in the 3 mutants, they provide a qualitative illustration of the reaction pathway

3

.differences between them. Only the percentage of the yield is labeled in the reaction schemes, to

4

highlight the branching ratio for the reactions from the P+I– state. The reaction rate of each path

5

can in principle be calculated using the equation τi=φiτi, obs, where φi is the relative amount of P+

6

in the DAS with the observed lifetime of τi, obs.

7

The branching ratio between the forward electron transfer reaction P+I– → P+QA and P+I–

8

recombination may be calculated from the relative amplitudes of the 870-nm band in the

9

different DAS. As shown in Figure 5, the major reaction pathways for M214LH and M214LQ

10

are the same, with only yield differences. The amount of P+ remaining in the P+QA– state and that

11

lost due to P+I– recombination are the values at 870 nm in the longest-lived DAS and the sum of

12

the values from the 220-ps and the 800/900-ps DAS, respectively. However, the reactions in the

13

M214LN mutant are quite different from the other two mutants. First, the amount of P+ in the

14

non-decaying DAS of M214LN is much higher than in the other two mutants and the lifetime is

15

much longer than the longest-lived components in M214LH and M214LQ. Second, a significant

16

amount of P+I– (about 45%) is contained in the non-decaying DAS, in addition to the P+QA–

17

state. A similar scenario was observed in the study of a set of non-β-mutants with the amino acid

18

at M214 position varied 41. In that study, an energetically relaxed P+HA– state, denoted (P+HA–)f,

19

with a much longer lived lifetime than that of the initially formed P+HA– state, was proposed. A

20

similar relaxed state, (P+I–)', is proposed here in the reaction scheme for the M214LN mutant

21

(Figure 5, LN).

22 23

Conclusions 21


Biochemistry

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Page 22 of 36

1

Past studies of mutations at M214 have shown that side chain volume, and by inference the

2

flexibility of the protein, are key factors in controlling the fate of the initial charge separated

3

state 41. In that work, a systematic progression in sidechain volume from methionine (similar in

4

size to the original leucine residue), to smaller residues (in the order cysteine, alanine, glycine)

5

resulted in increasing kinetic heterogeneity and associated long-lived P+HA– (in these mutants,

6

HA remains a bacteriopheophytin). A dynamic model developed for the system was consistent

7

with the idea that decreasing the volume of the M214 amino acid side chain increases the

8

flexibility near the cofactor HA enabling fast energetic relaxation of the HA– anion radical. This

9

results in slower electron transfer from HA– to QA and the long-lived P+HA– states. In the current

10

work, the situation is more complex; both the side chain volume at M214 and the electronic

11

properties of the cofactors themselves are being altered by the mutations. Previous work has

12

shown that introducing a ligand at M214 changes the cofactor at HA from bacteriopheophytin to

13

bacteriochlorophyll and alters the free energy of the resulting P+β– state so that it approaches that

14

of P+BA– and P*. This modification is associated with an order of magnitude increase in the rate

15

of P+β– recombination relative to P+HA– in wild type 28, 29, 33, 37. In this respect, the comparison of

16

the mutants M214LH, M214LQ and M214LN is interesting. All three mutants result in the

17

conversion of the bacteriopheophytin on the A-side to a bacteriochlorophyll. Thus at early times

18

they all show the formation of an apparent equilibrium mixture of P+β– and P+BA– (i.e., P+I–).

19

However, while the H and Q substitutions show quite similar kinetic decay profiles of P+I–, a

20

very different decay profile is seen for M214LN (Figure 3C-D). The only structural difference

21

between N and Q is a shorter length of the side change by one methylene group in case of N. H

22

has a volume very similar to Q, but a very different electronic structure from either Q or N. As

23

in the earlier studies 41, one observes quite heterogeneous kinetics of P+I– decay including a long22


Page 23 of 36

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Biochemistry

1

lived species. In analogy to previous work, it is likely that substitution at M214 with the smaller

2

asparagine provides additional flexibility in the region allowing relaxation of P+I– in competition

3

with the formation of P+QA–. This relaxed P+I– state, however, does not significantly contribute

4

to forward electron transfer, resulting in a fraction (~30%) of the P+I– state lasting for the

5

duration of the measured time course, similar to that seen in the M214LG mutant in the

6

previously studied series of the M214 mutants 41. Apparently, for the P+I– → P+QA– reaction, the

7

amino acid side chain volume has a more profound effect than does the more obvious electronic

8

properties of the side chain: the histidine contains an aromatic imidazole group quite different

9

from both glutamine and asparagine, yet the M214LH and M214LQ are much more similar to

10

each other in terms of the rate of electron transfer to the quinone than either is to M214LN.

11 12

This relaxation also appears to be the reason why the long-lived stimulated emission seen in

13

M214LH and M214LQ are not present in M214LN (Fig 2A). Long-lived stimulated emission

14

presumably comes about because P+I– is elevated in free energy in M214LH and M214LQ

15

relative to P+HA– in the wild type. However, the rapid relaxation of P+I– in the M214LN mutant

16

presumably makes P* inaccessible.

23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

AUTHOR INFORMATION

2

Corresponding Author

3

*Neal W. Woodbury ([email protected])

Page 24 of 36

4 5

Author Contributions

6

R. Saer designed and made reaction center mutants; J. Pan carried out laser spectroscopic

7

measurements; J. Pan and S. Lin performed data analysis and kinetic modeling; J. T. Beatty and

8

N. W. Woodbury supervised the work and edited the manuscript. All authors have given

9

approval to the final version of the manuscript.

10 11

Acknowledgements

12

This work was funded by NSF grants MCB-0642260 and MCB-1157788 at ASU. JTB thanks

13

NSERC Canada for funding through the Discovery Grants system; RGS thanks NSERC for a

14

postgraduate fellowship.

15 16 17 18 19 20

Authors Jie Pan, Su Lin, and Neal W. Woodbury received funding from NSF grants MCB0642260 and MCB-1157788. Author J. Thomas Beatty received funding from Canadian Natural Sciences and Engineering Research Council Discovery Grant 2796. Author Rafael Saer was supported by a postgraduate fellowship from the Canadian Natural Sciences and Engineering Research Council. 24


Page 25 of 36

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Figures A

B Absorbance (a.u.)

1

WT LH LQ LN B/P

x2

H H

∆A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

x2

B

P LQx2

500 550 600 750 800 850 900 Wavelength (nm)

25

Figure 1. A, Structure of the wild type Rb. sphaeroides RC cofactors (PDB code: 2J8C) with

26

cofactors labeled (P, BA, BB, HA, HB, QA, QB). The subscripts A and B denote the cofactors in the

27

A- and B-branch, respectively. The position of leucine (L) M214 in the structure of wild type

28

RCs is shown as meshed ball structure. Forward electron transfer pathways (P → BA → HA →

29

QA → QB) and associated time constants are indicated by bold arrows and text, respectively. The

30

charge recombination of P+HA– is indicated by the curved blue arrow. B, Upper panel: room

31

temperature absorption spectra of wild type (black), M214LH (dark cyan), M214LQ (blue) and

32

M214LN (red) mutant RCs with background subtracted. Spectra were scaled to have the same

33

absorbance at 865 nm, and a straight line from 460 to 640 nm was subtracted from the QX region

34

spectra and from 700 and 940 in the QY region spectra. Assignments of bands of P, B and H in 25


Biochemistry

1

both QX and QY regions are indicated for wild type RCs. B, Lower panel: difference absorption

2

spectra between the mutants and wild type, obtained by subtracting the wild type absorption

3

spectrum from the corresponding mutant. All spectra in the QX region, and the difference

4

spectrum of M214LN (M214LN minus wild type) in QY region are multiplied by a factor of 2, as

5

is the difference spectrum of M214LQ.

6 7

A 920 nm ∆A (a.u.)

0.0 0.0

-0.5

0.0 -0.5 -0.1 -1.0

-1.0

0

0 10 20 10

∆A (mOD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

10

20 ps 0

2 ns

1

100 Time(ps)

1000

B 25 ps

0 5

-10 500

WT LH LQ LN

550

0

500 fs

-5 500

600

600 650 Wavelength (nm)

700

700

8 9 10

Figure 2. A, Normalized kinetic traces of RC samples: wild type (black); M214LH (dark cyan);

11

M214LQ (blue); and M214LN (red). Excitation at 865 nm, and probe at 920 nm. The smooth

12

curves represent double exponential fitting with parameters listed in table I. The time scale is 26


Page 27 of 36

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Biochemistry

1

linear before the axis break and logarithmic thereafter. Insets: same kinetics shown in the main

2

panel (left panel) normalized at bleaching maxima and set to zero at 25-ps delay to compare the

3

early decay phase, (right panel) plotted on a linear time scale and at a reduced Y-axis to compare

4

the later decay kinetics. B, Transient absorption spectra in the QX region at 25 ps time delay.

5

Data points are shown as symbols with a smooth curve drawn through the points, and the same

6

color code as in panel A. The negative bands at 595 nm and 540 nm in the WT difference

7

spectrum are due to the ground state bleaching of P and HA (a BPhe molecule), respectively. The

8

absorption increase peaking at 665 nm is due to the absorption of the HA– anion radical. In the β-

9

mutants, the bleaching at 595 nm is due to ground state bleaching of P and I (mix of BA and β).

10

The absorption increases in the 620 to 700 nm region are the spectral features associated with the

11

I– anion (620 – 680 nm) and the P+ cation band at 700 nm. Inset, transient absorption spectra at

12

500-fs time delay. The curves for all mutants are scaled in such a way that the bleaching

13

maximum of the QX transition near 600 nm at 500 fs is the same as that in wild type.

27


Biochemistry

∆A (mOD)

5

20

0

0

-5

-10 500

1.0

WT M214LH M214LQ M214LN 550 600 550 600 650 Wavelength (nm)

-20 650

700

700

700

750

800

850

900

-40 950

Wavelength (nm)

D

C

0.0

830 nm -0.5

0.5

665 nm (WT) 635 nm (LH,LQ,LN)

0.0 0

5 10

100 Time(ps)

-1.0

WT

1000

0

5 10

∆ A (a.u.)

∆A (a.u.)

B Qy

A Qx

∆ A (mOD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

100

1000

Time(ps)

3

1 2 Figure 3. Transient absorption spectra in the (A) QX and (B) QY transitions at 4 ns after 865-nm

4

excitation of RC samples: wild type (black); M214LH (dark cyan); M214LQ (blue); M214LN

5

(red). All time resolved spectra are uniformly scaled in the same way as in Figure 2B. Data

6

points are shown as symbols with a smooth curve drawn through the points. For WT, M214LH

7

and M214LQ, the absorption decrease at 600 nm and the small increase around 700 nm in A, as

8

well as the bleaching at 865 nm and the derivative-shaped spectral changes in the 750 – 810 nm

9

region in B, are due to P+ in the QX and QY region, respectively. The spectral features of

10

M214LN are dominated by the P+I- state. The inset in A compares the time-resolved spectra from

11

the three mutants normalized at the bleaching of the 600-nm band at 4-ns time delay. C, kinetic

12

traces at 665 nm for the wild type RC and 635 nm for the three β–mutants. D, kinetic traces at

13

830 nm for the same set of samples. Curves are normalized at the maxima. The smooth curves 28


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Biochemistry

1

represent multiple exponential fitting. The time axis is linear until 10 ps and then logarithmic to

2

6 ns. All color-codes are the same as in panel A.

3

29


Biochemistry

∆A (mOD)

10

A WT

0

2.7 ps 9.8 ps

-10

∆A (mOD)

10

3.2 ps 15.0 ps

-10 10

D LQ

C LN

0

0

2.7 ps 12.0 ps

-10 600

700

2.9 ps 14.0 ps 800

900

600

∆A (mOD)

10

E WT

700

-10 800

900

Wavelength (nm)

Wavelength (nm)

1

5

F LH

0 -10 -20 5

0

180 ps 800 ps 10.8 ns

220 ps 1.0 ns ND

-5 -10

G LN

H LQ

0 -5 -10

0

220 ps 2.6 ns ND 600

2 3

10

B LH

0

∆A (mOD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

700

230 ps 900 ps 15.3 ns 800

900

Wavelength (nm)

600

700

-5

800

900

Wavelength (nm)

4

Figure 4. Decay associated spectra (DAS) obtained from 5-exponential fitting for the 2 fast

5

components of: A, wild type; B, M214LH; C, M214LN; D, M214LQ; and E to H for the 3 long-

6

lived DAS. The QX (500 to 780 nm) and QY (730 to 980 nm) transition data were fit separately 30


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Biochemistry

1

using the same set of decay components and plotted directly without further scaling. Lifetimes of

2

each DAS are listed in the legend. Non-decaying components were defined if the lifetime is more

3

than 10 times longer than the measuring time window of 6 ns. The lifetimes of the non-decaying

4

components of the WT and M214LN mutant are 1.11 and 10.8 µs, respectively.

5 6 7

8 9 10

Figure 5. Reaction scheme of wild type and 3 β-mutant RCs. The relative yields along each of

11

the pathways are calculated based on a 5-exponential fitting results shown in Figure 4 and Figure

12

S2. See text for details.

13

31


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

1

Table 1. P* decay lifetimes obtained from global fitting of the transient absorption spectra in the

2

QY transition wavelength region using 5 kinetic components. The average lifetimes, τave, were

3

calculated using the following equation.

=

∑ ∑

4

Where Ai and τi are the amplitude at 900 nm and the lifetime components of the two shortest-

5

lived DAS.

6 RC Samples

P* (λ = 900 nm)

P* ave from Kirmaier

P* from Saer

τ1 (ps), A1 %

τ2 (ps), A2%

τave (ps)

τ (ps)

τ (ps)

Wild Type

2.7 ps, 81%

9.8 ps, 19%

4.0

3.5+0.3

3.8+0.2

M214LH

3.2 ps, 68%

15.0 ps, 32%

6.9

6.5+0.8

4.8+0.5

M214LQ

2.9 ps, 68%

14.0 ps, 32%

6.5

NA

NA

M214LN

2.7 ps, 63%

12.0 ps, 37%

6.1

NA

NA

7 8 9 10 11 12 13 14 15 16

32


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Biochemistry

1

ASSOCIATED CONTENT

2 3 4 5

Normalized transient absorption spectra at 25 ps and 4 ns, the three long-lived DAS obtained from 5-exponential fitting for all RC samples, and the kinetic models with calculated electron transfer rates are given in the Supporting Information.

6

References

7 8 9

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TOC

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