Skip to main content
SpringerLink
Log in

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired Photosystem II photochemical activity

  • Research Article
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

Chl:

Chlorophyll

DCMU:

3-(3,4-Dichlorophenyl)-1,1-dimethylurea

F:

The fluorescence level at time t

(F-F0)/F0 :

Fluorescence yield at a particular time

F0 :

Fluorescence emitted by a sample at low-light levels prior to actinic-flash excitation

Fi :

The initial peak of a fluorescence induction curve

Fmax :

Maximum fluorescence yield following actinic-flash excitation

F′m :

Maximum fluorescence level under the ambient light induced by a saturating light pulse

Fp :

The saturating level of a fluorescence induction curve

Ft :

The steady-state level of fluorescence measured under ambient light prior to a saturating light pulse

\(\frac{\Delta F} {F^{\prime}_m}=(F^{\prime}_{m}-F_i)/F^{\prime}_{m}\) :

Measure of the photochemical conversion efficiency (or photochemical activity) of PSII

Fv :

Variable fluorescence level (Fm−F0)

OEC:

O2-Evolving complex

PAM:

Pulse amplitude modulated

PAR:

Photosynthetically active radiation

PQ:

Plastoquinone

PSII:

Photosystem II

pWt:

Pseudo-wild-type

QA :

The primary quinone acceptor of PSII

QB :

The secondary quinone acceptor of PSII

TAP:

TRIS-acetate-phosphate medium

YD :

Tyr-160 of the D2 polypeptide

References

  • Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV, Ghirardi ML, Rubin AB, Tsygankov AA, Seibert M (2003) The dependence of algal H2 production on Photosystem II and O2 consumption activities in sulfur deprived Chlamydomonas reinhardtii cells. Biochim Biophys Acta 1607:153–160

    Article  CAS  PubMed  Google Scholar 

  • Bricker TM, Lowrance J, Sutton H, Frankel LK (2001) Alterations of the oxygen-evolving apparatus in a (448)Arg → (448)S mutant in the CP47 protein of photosystem II under normal and low chloride conditions. Biochemistry 40:11483–11489

    Article  CAS  PubMed  Google Scholar 

  • Cao JC, Vermaas WF, Govindjee (1991) Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in photosystem II of Synechocystis sp. PCC Biochim Biophys Acta 1059:171–180

    Article  CAS  Google Scholar 

  • Chu HA, Nguyen AP, Debus RJ (1994) Site-directed photosystem II mutants with perturbed oxygen-evolving properties 1. Instability or inefficient assembly of the manganese cluster in vivo. Biochemistry 33:6137–6149

    Article  CAS  PubMed  Google Scholar 

  • Chu HA, Nguyen AP, Debus RJ (1995) Amino acid residues that influence the binding of manganese or calcium to photosystem II. 1. The carboxy-terminal domain of the D1 polypeptide. Biochemistry 34:5839–5858

    Article  CAS  PubMed  Google Scholar 

  • Flynn T, Ghirardi ML, Seibert M (2002) Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection. Int J Hydrogen Res 27:1421–1430

    Article  CAS  Google Scholar 

  • Forestier M, King P, Zhang L, Posewitz M, Schwarzer S, Happe T, Ghirardi ML, Seibert M (2003) Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur J Biochem 270:2750–2758

    Article  CAS  PubMed  Google Scholar 

  • Gfeller RP, Gibbs M (1984) Fermentative metabolism of Chlamydomonas reinhardtii, I: Analysis of fermentative products from starch in dark and light. Plant Physiol 75:212–218

    CAS  PubMed  Google Scholar 

  • Ghirardi ML, Lutton TW, Seibert M (1996) Interactions between diphenylcarbazide, zinc, cobalt, and manganese on the oxidizing side of Photosystem II. Biochemistry 35:1820–1828

    Article  CAS  PubMed  Google Scholar 

  • Ghirardi ML, Togasaki RK, Seibert M (1997) Oxygen sensitivity of algal H2-production. Appl Biochem Biotech 63:141–151

    Google Scholar 

  • Ghirardi ML, Lutton TW, Seibert M (1998) Effects of carboxyl amino acid modification on the properties of the high-affinity, manganese-binding site in Photosystem II. Biochemistry 37:13559–13566

    Article  CAS  PubMed  Google Scholar 

  • Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, Greenbaum E, Melis A (2000) Microalgae: a green source of renewable H2. Trends Biotechnol 18:506–511

    Article  CAS  PubMed  Google Scholar 

  • Ghirardi ML, King P, Kosourov S, Forestier M, Zhang L, Seibert M (2005) Development of algal systems for hydrogen photoproduction: addressing the hydrogenase oxygen-sensitivity problem. In: Collings AF, Critchley C (eds) Artificial photosynthesis: from basic biology to industrial application. Wiley-VCH Verlag, Weinheim Germany, pp 213–227

    Google Scholar 

  • Ghirardi ML, Posewitz MC, Maness PC, Dubini A, Yu Y, Seibert M (2007) Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms. Ann Rev Plant Biol 58:71–91

    Article  CAS  Google Scholar 

  • Guenther JE, Nemson JA, Melis A (1990) Development of PSII in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PSIIβ, QB-non-reducing centers to the PSIIα, Qb-reducing form. Photosynth Res 24:35–46

    Article  CAS  Google Scholar 

  • Happe T, Kaminski A (2002) Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur J Biochem 269:1022–1032

    Article  CAS  PubMed  Google Scholar 

  • Happe T, Hemschemeier A, Winkler M, Kaminski A (2002) Hydrogenases in green algae: do they save the algae’s life and solve our energy problems? Trends Plant Sci 7:246–250

    Article  CAS  PubMed  Google Scholar 

  • Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. p 780. Academic Press, San Diego

  • Kosourov S, Tsygankov A, Seibert M, Ghirardi ML (2002) Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: effects of culture parameters. Biotech Bioeng 78:731–740

    Article  CAS  Google Scholar 

  • Kosourov S, Seibert M, Ghirardi ML (2003) Effects of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant Cell Physiol 44:146–155

    Article  CAS  PubMed  Google Scholar 

  • Kosourov S, Makarova V, Fedorov AS, Tsygankov A, Seibert M, Ghirardi M (2005) The effect of sulfur re-addition on H2 photoproduction by sulfur-deprived green algae. Photosynth Res 85:295–305

    Article  CAS  PubMed  Google Scholar 

  • Kosourov S, Patrusheva E, Ghirardi ML, Seibert M, Tsygankov A (2007) A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions. J Biotechnol 128:776–787

    Article  CAS  PubMed  Google Scholar 

  • Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42:313–349

    Article  CAS  Google Scholar 

  • Kreutzberg K (1984) Starch fermentation via a formate producing pathway in Chlamydomonas reinhardtii, Chlorogonium elongatum and Chlorella fusca. Physiol Plantarum 61:87–94

    Article  Google Scholar 

  • Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382

    Article  CAS  PubMed  Google Scholar 

  • Lazár D (1999) Chlorophyll a fluorescence induction. Biochim Biophys Acta 1412:1–28

    Article  PubMed  Google Scholar 

  • Li Z, Burnap RL (2002) Mutations of basic arginine residue 334 in the D1 protein of Photosystem II lead to unusual S2 state properties in Synechocystis sp. PCC 6803. Photosynth Res 72:191–201

    Article  CAS  PubMed  Google Scholar 

  • Loll B, Kern J, Saenger W, Zouni A, Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II.Nature 438:1040–1044

    Article  CAS  PubMed  Google Scholar 

  • Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122:127–136

    Article  CAS  PubMed  Google Scholar 

  • Minagawa J, Crofts AR (1994) A robust protocol for site-directed mutagenesis of the D1 protein in Chlamydomonas reinhardtii: a PCR spliced psbA gene in a plasmid conferring spectinomycin resistance was introduced into a psbA deletion strain. Photosynth Res 42:121–131

    Article  CAS  Google Scholar 

  • Nixon PJ, Diner BA (1992a) Aspartate 170 of the photosystem II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. Biochemistry 31:942–948

    Article  CAS  PubMed  Google Scholar 

  • Nixon PJ, Trost JT, Diner BA (1992b) Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in Photosystem II of the cyanobacterium Synechocystis sp PCC 6803: assembly requires a free carboxyl group at C-terminal position 344. Biochemistry 31:10859–10871

    Article  CAS  PubMed  Google Scholar 

  • Ohta S, Miyamoto K, Miura Y (1987) Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation. Plant Physiol 83:1022–1026

    Article  CAS  PubMed  Google Scholar 

  • Posewitz MC, King PW, Smolinski SL, Zhang L, Seibert M, Ghirardi ML (2004) Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe]-hydrogenase. J Biol Chem 279:25711–25720

    Article  CAS  PubMed  Google Scholar 

  • Ruffle SV, Wang J, Johnston HG, Gustafson TL, Hutchison RS, Minagawa J, Croffts A, Sayre RT (2001) Photosystem II peripheral accessory chlorophyll mutants in Chlamydomonas reinhardtii. Biochem character sensitivity photo-inhibit Plant Physiol 127:633–644

    CAS  Google Scholar 

  • Schreiber U, Hormann H, Neubauer C, Klughammer C (1995) Assessment of Photosystem II photochemical quantum yield by chlorophyll fluorescence quenching analysis. Plant Physiol 22:209–220

    CAS  Google Scholar 

  • Semin B, Parak F (1997) Coordination sphere and structure of the Mn cluster of the oxygen-evolving complex in photosynthetic organisms. FEBS Letters 400:259–262

    Article  CAS  PubMed  Google Scholar 

  • Svensson B, Vass I, Styring S (1991) Sequence analysis of the D1 and D2 reaction center proteins of photosystem II Z.Naturforsch 46:765–776

    Google Scholar 

  • Tsygankov A, Kosourov S, Seibert M, Ghirardi ML (2002) Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures. Int J Hydr Energy 27:1239–1244

    Article  CAS  Google Scholar 

  • Tsygankov AA, Kosourov SN, Tolstygina IV, Ghirardi ML, Seibert M (2006) Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int J Hydrogen Energy 31:1574–1584

    Article  CAS  Google Scholar 

  • Wykoff DD, Davies JP, Melis A, Grossman AR (1998) The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physiol 117:129–139

    Article  CAS  PubMed  Google Scholar 

  • Xiong J, Hutchison RS, Sayre RT, Govindjee (1997) Modification of the photosystem II acceptor side function in a D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardti. Biochim Biophys Acta 1322:60–76

    Article  CAS  PubMed  Google Scholar 

  • Xiong J, Minagawa J, Crofts A, Govindjee (1998) Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii. Biochim Biophys Acta 1365:473–491

    Article  CAS  PubMed  Google Scholar 

  • Young A, McChargue M, Frankel LK, Bricker TM, Putnam-Evans C (2002) Alterations of the oxygen-evolving apparatus by a 305→305 Ser mutation in the CP43 protein of Photosystem II from Synechocystis sp. PCC 6803 under chloride-limiting conditions. Biochemistry 41:15747–15753

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Happe T, Melis A (2002) Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green algae). Planta 214:552–561

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge the NREL LDRD program (MS), the Division of Energy Biosciences, Basic Energy Sciences, US Department of Energy (MS and MLG), and the Russian Foundation for Basic Research (ABR, TEK, BKS, GPK and SK). The work at the Ohio State University was supported under subcontract through NREL (RTS). Valeria Makarova would also like to thank the NREL LDRD program for support while at NREL.

Author information

Author notes

  1. Valeria V. Makarova

    Present address: California Lutheran University, 60 West Olsen Rd, Thousand Oaks, CA, 91360, USA

Authors and Affiliations

  1. National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO, 80401, USA

    Valeria V. Makarova, Sergey Kosourov, Maria L. Ghirardi & Michael Seibert

  2. Moscow State University, Leninskie Gori, Moscow, 119899, Russia

    Valeria V. Makarova, Tatiana E. Krendeleva, Boris K. Semin, Galina P. Kukarskikh & Andrei B. Rubin

  3. Institute of Basic Biological Problems, RAS, Pushchino, Moscow Region, 142290, Russia

    Sergey Kosourov

  4. Ohio State University, Columbus, OH, 43210, USA

    Richard T. Sayre

Authors
  1. Valeria V. Makarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sergey Kosourov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatiana E. Krendeleva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Boris K. Semin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Galina P. Kukarskikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrei B. Rubin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Richard T. Sayre
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Maria L. Ghirardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Seibert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Seibert.

Appendix

Appendix

Fig. 5
figure 5

EPR spectrum of pWt cells sampled during the mid-logarithmic phase of growth. The amount of assembled PSII complex was determined from EPR measurements of the SII signal performed with a Bruker E500 spectrometer at low temperature (77 K). All samples exhibited a narrow (line width, ΔH = 9–12 Gauss) EPR signal at g = 2.0041, attributable to presence of the Y +D paramagnetic species. No significant difference was found in the EPR signal parameters or the concentration of Y +D spins, calculated on a Chl basis, between the pWt and any of the R323 mutants. The EPR signal intensities of the mutants normalized per mg of Chl varied within 20% of the pWt signal, which is shown above. Sample size, 0.3–0.4 ml; total Chl concentration, 1.5–2 mg ml−1. EPR recording conditions: frequency, 9.4 GHz; power, 6 mW; field modulation amplitude 1G; field modulation frequency, 100 kHz; single scan. The spectrum was recorded in dark

Full size image
Fig. 6
figure 6

Chl fluorescence induction curves for pWt and mutant algal culture samples grown in sulfur-replete TAP medium. All curves were measured with a PAM fluorometer. The samples were removed from the bioreactors during the mid-logarithmic phase of growth, adjusted to equal Chl concentration, and dark adapted for 2 min before measurements. The fluorescence induction curve of the control pWt sample exhibited two components. The fast component (from F0 to Fi) reflects the accumulation of reduced QA in QB-non-reducing PSII centers and the presence of PSII centers with a doubly-reduced QB prior to illumination. The slow component (from Fi to Fp) represents the subsequent accumulation of reductants in the PQ-pool at high light intensity. The induction curves measured with the R323H and R323N mutants had shapes similar to that of pWt. However, the F0 level was increased, and the maximal fluorescence yield was reduced compared to the pWt. The R323D curve had an intermediate character. The fluorescence induction curve measured with the R323E mutant exhibited a barely-detectable, residual, slow-rise component. The halftime for transition from the Fi to the Fp level (in ms), which is a measure of the time it takes to reduce the PQ pool, also increased progressively in the mutants: pWt (170), R323H (240), R323N (270), and R323D (390)

Full size image
Fig. 7
figure 7

Flash-probe chlorophyll fluorescence decay kinetics of pWt and R323H,N,D,E cells in the presence of DCMU (which blocks Q- A → QB electron transfer). Samples were taken from the bioreactors during the mid-logarithmic phase of growth on TAP medium, concentrated by centrifugation to a Chl concentration of 40 μg ml−1, and dark adapted for 5 min before measurement

Full size image
Table 3 Flash-induced fluorescence decay after a single flash
Full size table

Rights and permissions

Reprints and permissions

About this article

Cite this article

Makarova, V.V., Kosourov, S., Krendeleva, T.E. et al. Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired Photosystem II photochemical activity. Photosynth Res 94, 79–89 (2007). https://doi.org/10.1007/s11120-007-9219-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-007-9219-4

Keywords

Search

Navigation