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Technical Communication

Arginine171 of Chlamydomonas reinhardtii [FeeFe]hydrogenase HydA1 plays a crucial role in electrontransfer to its catalytic center

Kateryna Sybirna a, Pierre Ezanno b,1, Carole Baffert b, Christophe Leger b, Herve Bottin a,*a IBiTec-S, SB2SM, LMB (UMR CNRS 8221), DSV, CEA, 91191 Gif-sur-Yvette, FrancebCNRS, Aix Marseille Universite, BIP UMR 7281, IMM FR 3479, 13402 Marseille, France

a r t i c l e i n f o

Article history:

Received 17 September 2012

Received in revised form

3 December 2012

Accepted 11 December 2012

Available online 18 January 2013

Keywords:

[FeeFe] hydrogenases

Ferredoxin-binding site

Hydrogen evolution

Catalytic activity

* Correspondingauthor. CEAdeSaclay,DSV, IBiE-mail address: herve.bottin@cea.fr (H. B

1 Present address: Institute of Biophysics,0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2012.12.0

a b s t r a c t

[FeeFe] hydrogenases, with hydrogen evolution activities outperforming [NieFe] hydrog-

enases by 3e4 orders of magnitude, are still the most promising enzyme class for hydrogen

production purposes. For Chlamydomonas reinhardtii [FeeFe] hydrogenase HydA1 the

question of catalytic activity and electron transport is of main importance. Here we report

the characterization of two mutant forms of C. reinhardtii HydA1. An aspartic acid in place

of arginine171 leads to a six-fold increase of the catalytic activity in comparison to the wild

type protein during methyl viologen-dependent hydrogen production. Tryptophan in

position 171 does not result in any change in methyl viologen-induced activity. At the same

time these mutations lead to a strong decrease in ferredoxin-dependent hydrogen

production while the catalytic center of mutant forms stays intact. The localization of

this amino acid (arginine171) in the environment of CrHydA1 H-cluster indicates that the

limitation of the catalytic activity of this hydrogenase is due to the electron transfer step to

the catalytic center where the reduction of protons takes place.

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction partners, there is the [FeeFe] hydrogenase HydA1 [4]. This

Chlamydomonas reinhardtii is a photosynthetic microalgae

capable of hydrogen production under anaerobiosis [1]. In the

chloroplasts of C. reinhardtii after the reduction of the low

potential soluble electron carrier ferredoxin (PetF or Fd) by the

membrane protein photosystem I (PS I), Fd transfers its electron

to a number of partners that are involved in many metabolic

processes such as sulfur and nitrogen assimilation, reduction

of NADPþ, regulation of the Calvin cycle, cyclic electron

transfer and dihydrogen production [2,3]. Among ferredoxin

Tec-S, SB2SM(UMRCNRS8ottin).Chinese Academy of Scie2012, Hydrogen Energy P78

enzymereversibly catalyzes the reaction:H24 2Hþþ 2e�, usingelectrons provided by reduced Fd (Fdred), according to the

reactions:

2Fdred þ 2Hþ / 2Fdox þ H2

[FeeFe] hydrogenases are the metalloenzymes that show

the highest catalytic activity in the hydrogen production [5].

Thismakes themparticularly interesting for sustainable clean

energy production.

221), LMB,91191Gif surYvetteCedex, France.Tel.:þ33 (0) 169089868.

nces, 100101 Beijing, Pekin, China.ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 2 9 9 8e3 0 0 2 2999

C. reinhardtii possesses two monomeric [FeeFe] hydroge-

nases: HydA1 and HydA2 [6]. The active site of HydA1, which

receives electrons from PetF, is called the H-cluster [7]. It

consists of a dinuclear iron center and a [4Fee4S] cluster.

Unlike bacterial [FeeFe] hydrogenases, it is the only FeeS

cluster in this enzyme.

Recently,Winkler and co-authors demonstrated that Lys396

of HydA1, which is particularly conserved among green algal

hydrogenases, is crucial for a successful binding and electron

transfer between PetF and HydA1 [8]. In this work we dem-

onstrate the occurrence of another positively charged amino

acid close to the CrHydA1 surface and conserved among

green algal hydrogenases but not conserved among [FeeFe]

hydrogenases of bacterial type (Arg171 e not counting the first

56 amino acids of the transit peptide which is not included in

this construction [9]) to be essential for the successful binding

and electron transfer between ferredoxin and HydA1. Its non-

conservation among [FeeFe] hydrogenases of bacterial type

points to its importance to algae-specific electron transfer

reaction.

2. Materials and methods

2.1. Mutagenesis and purification of CrHydA1

Site-directed mutagenesis was performed on the CrhydA1

gene cloned into the pBBR-hydA1N vector [10] using Quik-

Change Site-Directed Mutagenesis Kit (Agilent Technologies,

Santa Clara, CA, USA). The recombinant plasmid was intro-

duced and expressed in Shewanella oneidensis AS52 strain as

described in Ref. [10]. An average amount of 0.4 mg of purified

active protein per liter of culture was obtained for both native

and mutant enzymes. Enzyme purification and activity assay

were both performed in a glove box (Jacomex) filled with N2.

2.2. Hydrogen evolution assay

Hydrogen formation assay on purified HydA1 and mutant

forms were performed in a glove box (Jacomex) filled with N2,

as described previously in Ref. [10]. Determinations of Vmax

and Km for in vitro H2 evolution were performed in the pres-

ence of either 0e6 mM reduced methyl viologen (MV) or

0e35 mM Synechocystis sp. PCC 6803 reduced ferredoxin in

50 mM Hepes, pH 6.7 at 20 �C. Ferredoxin was prepared as

described previously [11]. Hydrogen evolution was measured

by amperometry using a modified Clark-type electrode (Han-

satech, UK). The electrode was reversely polarized (þ0.7 V; Pt

vs Agþ/AgCl). The amplitude of the electrical signal from the

electrode was standardized using an aliquot of H2-saturated

solution as a reference.

2.3. Electrochemical experiments

Protein film electrochemistry experiments were carried out in

a glove box (Jacomex) filled with N2, using the electrochemical

setup and equipment (Autolab) previously described [12].

The two-compartment electrochemical cell was kept at the

desired temperature using a water circulation system. The

rotating disc pyrolytic graphite edge working electrode

(RDPGE) (area A z 1 mm2) was used in conjunction with an

electrode rotator, a platinum wire was used as a counter

electrode, and a saturated calomel electrode (SCE), located in

a side arm containing 0.1 M NaCl and maintained at room

temperature, was used as a reference. All potentials are

quoted versus the standard hydrogen electrode (SHE),

(ESHE ¼ ESCE þ 240 mV). The electrochemical cell contained

a buffer mixture of MES, CHES, TAPS, HEPES, and sodium

acetate (5 mM each), 1 mM EDTA and 0.1 M NaCl. The protein

films were prepared by painting the electrode with about half

a microliter of a stock enzyme solution (about 0.5 mM of

enzyme in the mixed buffer at pH 7 according to the enzyme

concentration).

3. Results and discussion

3.1. Localization and mutation of Arg171 in theenvironment of CrHydA1 H-cluster

The environment of CrHydA1 H-cluster shows the presence of

a positively charged amino acid, Arg171, which corresponds to

Arg187 (in pdb file 3LX4 [7]), that is non-conserved among

[FeeFe] hydrogenases of bacterial type and conserved among

green algal hydrogenases (Fig. 1A). This might indicate the

importance of this residue for partner recognition in green

algae. For example, in the hydrogenase of Clostridium aceto-

butylicum the residue corresponding to the arginine171 of C.

reinhardtii HydA1 is not a surface residue probably due to the

presence of the electron transfer domain. This residue is

absent (not necessary) in the bacterial hydrogenases (Fig. 1A)

because the electrons come from the above mentioned

domain. PetF from C. reinhardtii is a negatively charged protein

that transfers one electron to HydA1. The location of Arg171

between the H-cluster and the protein surface is shown in

Fig. 1B. The side chain of this residue is within 3.5e5.5�A of the

accessory [4Fee4S] sub-cluster using the partial HydA1

structure of Mulder and co-authors [7].

To study the contribution of Arg171 to the affinity between

hydrogenase and ferredoxin, we purified site-directedmutant

forms of C. reinhardtii HydA1 targeted at this position. The

positively charged amino acid (Arg171) was changed into

neutral and negatively charged residues (Trp for R171W and

Asp for R171D, respectively). It was expected that such

modifications would lower the affinity of ferredoxin for

hydrogenase.

3.2. Arg171 of HydA1 is involved in forming andstabilizing the electron transfer complex between ferredoxinand HydA1

Using reduced methyl viologen (MV$þ) as an artificial electron

donor, hydrogen evolution rates of about 700 mmol

H2 min�1 mg�1 protein, were measured for both the wild type

(WT) strain and R171W mutant (Table 1). In contrast, the

catalytic activity of R171D was about 6 times higher than the

activity of WT protein during MV-dependent H2 evolution

(Table 1). Contrary to MV-dependent H2 evolution, ferredoxin-

dependent evolution was significantly decreased by these

mutations. Changes of positively charged amino acid to

Fig. 1 e (A) Localization of Arg171 in the sequences of algal and bacterial [FeeFe] hydrogenases. Cr e Chlamydomonas

reinhardtii, So e Scenedesmus obliquus, Cf e Chlorella fusca, Ca e Clostridium acetobutylicum, Cp e Clostridium pasteurianum, Dd

e Desulfovibrio desulfuricans. (B) The location of Arg171 near the surface and the 4Fe4S sub-cluster of the apo hydrogenase

from C. reinhardtii (blue) (pdb 3LX4, [7]), this structure lacks the 2Fe sub-cluster, it houses only the 4Fe subpart of the H-

cluster. The structure was aligned with that of C. pasteurianum (pdb 3C8Y, [13]), whose complete H-cluster is also shown in

color (yellow [ S, orange [ Fe, blue [ N, white [ C, red [ O). The dotted line shows the shortest distance (3.5 A) between

the Nε nitrogen of Arg171 and the cubane.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 2 9 9 8e3 0 0 23000

neutral and negatively charged residues brought to the almost

complete loss of activity in HydA1/ferredoxin assay (Table 1).

This can be explained by the fact that Arg171 of HydA1 plays

a central role in guiding and binding the negatively charged

ferredoxin at the electron transfer site on HydA1. The effect of

electrostatic interactions on the binding between redox part-

ners has been largely documented: for example on the inter-

action between [NieFe] hydrogenase and cytochrome c3 in

Desulfovibrio vulgaris Miyazaki F [14] or on the binding of

photosystem I and ferredoxin in cyanobacteria [15].

Table 1 e Hydrogen evolution by HydA1 and mutant forms.

Strain Vmax (MV) mmol H2 mg�1 min�1 Km (MV) mM

WT 705 � 60 0.41 � 0.02

R171W 710 � 40 0.078 � 0.005

R171D 4600 � 240 1.2 � 0.1

Values are: mean result � standard deviation (at least three replicates o

surements were performed with 0.013e0.05 ng of hydrogenase in 50 mM

culations were performed by fitting the initial rates of hydrogen evolutio

The localization of this amino acid (Arg171) between the

H-cluster and the surface of CrHydA1 indicates that the

limitation of the catalytic activity of mutated hydrogenases

could result from the impaired step of electron transfer to the

catalytic center, where the reduction of the protons takes

place. The effect of the mutations on the electron transfer

promoted by reduced MV shows that the same Arg171 limits

the access of this molecule to the hydrogenase. The R171W

mutation (or removal of the positive charge) largely increases

the affinity for reduced MV without changing the maximum

H2 evolution Fd [34 mM] mmol H2 mg�1 min�1 (Fd [34 mM])

250 � 20

12.8 � 0.8

2.8 � 0.1

f all experiments). WT: wild type, MV: methyl viologen. In vitro mea-

Hepes pH 6.7, containing 20 mM Na-dithionite. Vmax and Km cal-

n to MichaeliseMenten kinetic equation.

Table 2eKinetic parameters of HydA1 andmutant forms.

CrHydA1(WT)

R171W R171D

Km (bar H2)a 1.3 � 0.2 1.3 � 0.2 1.1 � 0.2

kin CO (s�1 mM CO�1)b 75 � 10 70 � 10 70 � 10

kout CO (s�1)b 0.030 � 0.001 0.032 � 0.001 0.030 � 0.001

kapp(O2) (s�1 mM O2

�1)c 6 � 2 4 � 2 6 � 2

a The Km values were measured following the method described

in Ref. [21], at E ¼ �160 mV vs SHE.

b The kinetics constants relative to CO binding and release were

determined as described in Refs. [22,23] at E ¼ �160 mV vs SHE.

c kapp(O2) is the apparent bimolecular rate constant relative to the

reaction with O2 defined in Ref. [16] determined at E¼ 90mV vs SHE

for C. reihardtii HydA1 hydrogenase. All experiments were carried

out at 30 �C, pH 7, 1 bar H2, electrode rotation rate: 3 krpm.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 2 9 9 8e3 0 0 2 3001

rate of H2 evolution. An aspartic acid in position 171 results in

a very large increase of the Vmax of the enzyme (Table 1). This

demonstrates that electron transfer between the substrate

(Fdred or MV$þ) and HydA1 is largely controlled by electrostatic

interactions. It also shows that in the WT enzyme, the overall

H2 evolution is strongly limited by the electron transfer step

between HydA1 and the natural redox partner.

3.3. Protein film electrochemical experiments show thatthe cluster H is unaffected by the mutations

Having observed that mutations of Arg171 brought to almost

complete loss of ferredoxin-induced H2 evolution by HydA1

(Table 1) we wondered whether they also affect the properties

of the cluster H. The activity in presence of MV and the elec-

trochemical experiments show that in contrast to these

expectations, the catalytic center is not affected. WT CrHydA1

and the twomutants can be adsorbed at PGE electrode surface

and a catalytic current can be recorded both for H2 oxidation

and proton reduction (Fig. 2) [16].

In protein film voltammetry experiments, the magnitude

of the current is proportional to the turnover rate times of the

electroactive coverage and the latter cannot be measured and

may vary from one film to another. Therefore, the exact

magnitude of the signal should not be interpreted. However,

we have observed that the WT enzyme and the R171D and

R171W mutants give electrochemical signals whose magni-

tudes are very similar. In Fig. 2, these signals have been nor-

malized by the maximal value of the current. The positive

current at high potential is proportional to the rate of H2 oxi-

dation, whereas the negative current reveals H2 formation at

low potential. Overall, the three voltammograms exhibit

a very similar shape,with a decrease in H2 oxidation activity at

high potential that is due to oxidative anaerobic inactivation

[17]. However, the two mutants exhibit a greater H2 produc-

tion current than the WT enzyme, showing that when the

electrode replaces the soluble redox partner, both mutations

bias the enzyme in the reductive direction. We have

Fig. 2 e Voltammograms obtained with the WT (black),

R171D (red) and R171W (blue) enzymes adsorbed at

a pyrolytic graphite edge rotating disc electrode. The

current has been divided by the maximal current. Scan

rate: 20 mV/s, 30 �C, pH 7, 1 bar H2, electrode rotation rate:

3 krpm.

previously observed (and explained) that, in the case of the

[NieFe] hydrogenase from Desulfovibrio fructosovorans, point

mutations can strongly affect the ratio ofmaximal rates for H2

production versus H2 oxidation [18,19].

With the enzymes adsorbed onto graphite, we could use

themethodswe developed previously tomeasure a number of

enzymatic and kinetic parameters which report on the prop-

erties of the active site. Table 2 comparesWT CrHydA1 and the

two mutants (R171W and R171D) with respect to their

Michaelis constant for H2, the kinetics constants relative to CO

binding and release and the apparent bimolecular rate con-

stant relative to the reaction with O2 determined by cyclic

voltammetry [20]. In this respect, the properties of the three

enzymes are similar; demonstrating that the H-cluster is not

affected by the substitution of Arg171 to Trp or Asp.

4. Conclusions

In C. reinhardtii, electron transfer between ferredoxin and

the H-cluster of hydrogenase HydA1 occurs directly. Some

residues on the surface of hydrogenase play a central role in

guiding and binding ferredoxin at the electron transfer site of

HydA1. In this work we have demonstrated the presence

of positively charged amino acid on the surface of HydA1

(arginine171) to be essential for the efficient binding and elec-

tron transfer between Fd and HydA1. Mutation of this residue

results in the almost complete inhibition of electron transfer

between these protein partners without damaging of the

H-cluster. This study highlights the role of electrostatic

interactions, via Arg171, between HydA1 and Fd, the effect of

its mutation on electron transfer and most importantly, the

fact that electron transfer to the H-cluster is a limiting step in

H2 evolution. The understanding that in the WT enzyme H2

evolution is strongly limited by the electron transfer step will

help to create novel enzymes with improved catalytic activity

for hydrogen production purposes.

Acknowledgments

Our work was supported by the Agence Nationale de la

Recherche (AlgoH2).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 2 9 9 8e3 0 0 23002

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