The Presence of the IsiA-PSI Supercomplex Leads to Enhanced

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The Presence of the IsiA-PSI Supercomplex Leads to Enhanced Photosystem I Electron Throughput in Iron-Starved Cells of Synechococcus sp. PCC 7002 Junlei Sun, and John Harvey Golbeck J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b02176 • Publication Date (Web): 05 Jun 2015 Downloaded from http://pubs.acs.org on June 10, 2015

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The Journal of Physical Chemistry

The Presence of the IsiA-PSI Supercomplex Leads to Enhanced Photosystem I Electron Throughput in Iron-Starved Cells of Synechococcus sp. PCC 7002

Junlei Sun† and John H. Golbeck†,§,*

†

Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA

§

Dept. of Chemistry, The Pennsylvania State University, University Park, PA 16802 USA

* Corresponding author: John H. Golbeck, 328 South Frear Building, The Pennsylvania State University, University Park, PA 16802; phone: 814-865-1163; fax: 814-863-7024; email: [email protected]

Key Words: photosynthesis; photosystem I; phylloquinone; IsiA; IsiB; Fe-deficiency

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Abstract Photosystem I (PS I) is highly demanding of iron, requiring 12 atoms in the bound FX, FB, and FA iron-sulfur clusters and two atoms in the mobile acceptor protein ferredoxin. When grown under iron-limiting conditions, certain cyanobacteria express IsiA, a peripheral chlorophyll a antenna protein, and IsiB, a flavodoxin that substitutes for ferredoxin. The IsiA protein forms single and double rings around PS I, presumably to increase the optical cross-section so as to compensate for fewer PS I complexes. Previous studies have shown that IsiA serves as an efficient light-harvesting structure (Andrizhievskaya, G. G. et al. (2002) Biochim. Biophys. Acta 1556, 262-272), however, few, if any, studies have been carried out to show that the increased optical cross-section leads to an enhanced rate of electron transfer through PS I. Here, we report a more rapid transient accumulation of the A1– phyllosemiquinone anion radical by EPR spectroscopy in dark-adapted iron-depleted cells than in iron-replete cells after a block of white light. A derivative-shaped optical signal in the light-minus-dark difference spectrum of PS I from an electrochromic bandshift of a carotenoid located near the A1 phylloquinones is enhanced in iron-depleted wild-type cells and in an iron-depleted isiB deletion strain, which lacks flavodoxin, but is greatly diminished in an iron-depleted isiA deletion strain, which lacks IsiA and flavodoxin. These findings indicate that the transient accumulation of electrons on A1 occurs more rapidly in the IsiA/PS I supercomplex than in the PS I complex alone. Thus, the increased absorption cross-section from the IsiA proteins translates directly to an enhanced rate of electron transfer through PS I.

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Introduction Although iron is the fourth most abundant element in the Earth’s crust1, nearly all is present as the oxide due to the accumulation of an oxygen-containing atmosphere as a result of oxygenic photosynthesis2-3. Accordingly, the concentration of iron in surface ocean waters is low, typically between 0.02 nM and 1.0 nM4-5, making it one of the limiting elements in the growth of marine phytoplankton6. To cope with this amount of dissolved iron, alternative electron transfer proteins such as flavodoxin can substitute for ferredoxin7, making iron available to macromolecules for which it is indispensible8. In the mid-1980’s, studies on the Fe-starved unicellular cyanobacterium Anacystis nidulans R2 revealed the existence of an iron-stress-induced chlorophyll-binding protein9-12 in the CPVI-413-14 complex that was proposed to function as a light-harvesting antenna complex14. This protein was initially termed CP43’ because of its similarity to CP43 in PS II15 except that the former lacks a large hydrophilic loop that is present in the latter12. CP43’ was later identified as the product of the isiA gene in the isi (iron stress induced) operon16 of Synechococcus sp. PCC 7942, which also includes the downstream flavodoxin-encoding gene isiB17. The isi operon is transcriptionally regulated to be expressed under iron stress15, and although isiA and isiB are co-transcribed, the monocistronic message containing isiA is more abundant than the dicistronic message containing isiB15, 18. The isiA operon in Anabaena sp. PCC 7210 is further regulated by the product of the pkn22 gene19 and by IsrR, a cis-encoding antisense RNA transcribed from the isiA noncoding strand20-21. The product of the isiA gene is now commonly referred to as IsiA, although the term CP43’ is still used. In addition to Fe depletion, the isiA gene is induced by other

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stress conditions including oxidative stress

22-24

, salt-stress

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and heat stress 24. The IsiA

protein is expressed during the transition from exponential to the stationary growth state 25

, and apart from external stress conditions, certain mutations that block the electron

transfer from PS II to PS I also result in an up-regulation of the isiA gene 26-27. The function the IsiA protein was, at first, puzzling. A variety of functions were proposed including: i) IsiA functions as a Chl storage protein for the rapid recovery of cells from stressed conditions

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; ii) IsiA is an excitation energy dissipater that protects

PS II from photo-inhibition 29-32; iii) IsiA serves as a light-harvesting complex mainly for PS II

12, 14

or for both PS I and PS II

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; and iv) IsiA replaces CP43 of PS II under Fe-

deficient conditions and forms a cyclic electron transfer pathway around PS II and the cytochrome b6f complex 33-35. In the early 2000’s, two groups showed by electron microscopy (EM) and singleparticle analysis that under Fe-deficient conditions 18 copies of the IsiA protein form a ringed structure that completely surrounds trimeric PS I

36-38

. Based on analogy with

CP43, each IsiA protein was variously reported to contain 14-15 Chl a molecules39 and 16-17 Chl a molecules40, but more recent analysis of the absorption spectrum indicates that it could contain as few as 13 Chl a molecules41. Thus, the 18-mer ring surrounding trimeric PS I could increase the antenna size by 78 to 102%, depending on the number of Chl a molecules assigned to IsiA. The IsiA protein is remarkably flexible in its ability to bind to PS I. After prolonged growth in iron-deficient media, two complete rings surround trimeric PS I42, including 18 IsiA proteins in the inner ring and 25 IsiA proteins in the outer ring43. In a PsaF/PsaJ deletion mutant, the isiAB operon is induced27, and the trimeric PS I contains 17 IsiA proteins44, one less than the wild-type; and in a PsaL

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deletion mutant, which only contains monomeric PS I, partial single rings containing 6-7 IsiA proteins45 and partial double rings46 are found abutting the PsaF/PsaJ subunits. Severe iron stress of wild type leads to an increase in the amount of monomeric PS I47, which can then contain two complete rings with 12-14 IsiA proteins in the inner ring and 19-21 IsiA proteins in the outer ring48. Under iron stress, the amount of PS I (as well as the iron-rich Cyt b6/f complex) in Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 declines in abundance, leading to a decrease in the PS I/PS II ratio49. The implication is that the increase in the optical cross-section of the IsiA/PSI supercomplex compensates for the loss of PS I by absorbing, and processing, a greater number of photons per unit time. Indeed, temperature-dependent absorption and fluorescence studies of isolated IsiA/PSI supercomplexes have suggested that the IsiA ring increases the optical cross section by a factor of two40, and a kinetic study of isolated IsiA/PS I supercomplexes have shown longer equilibration and trapping times in the presence of IsiA, consistent with its function as an additional antenna for PS I50. Under long-term Fe starvation, IsiA accumulates in excess of what is needed, and IsiA ‘aggregates’ devoid of PS I can be observed42,

46, 48

. In the PsaF/PsaJ deletion

mutant, the isolated IsiA aggregates showed a fluorescence quantum yield of 2% compared to chlorophyll a in solution51, implying that the aggregates can protect cells against the harmful effect of light. Additional studies have confirmed that the IsiA aggregates function as efficient energy dissipators31,

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, thereby providing

photoprotection to the cell. The mechanism appears to involve transfer to a bound carotenoid54, which dissipates the energy by decaying to the ground state55.

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Thus far, most studies on the antenna function of IsiA-PS I supercomplexes have focused on fast excitation energy transfer and trapping processes, both of which occur on the

The Presence of the IsiA-PSI Supercomplex Leads to Enhanced

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