The cysteine-proximal aspartates in the Fx-binding niche of photosystem I. Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties

The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX.     

Control of electron transport in photosystem I by the iron-sulfur cluster FX in response to intra- and intersubunit interactions

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.     

The structure of genetically modified iron-sulfur cluster Fx in photosystem I as determined by X-ray absorption spectroscopy

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F_X, located on the core subunits PsaA/B to iron-sulfur clusters F_A/B on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F_X cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F_X was determined in PS I lacking clusters F_A/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F_X is discussed.     

Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

Iron-sulfur clusters in type I reaction centers.

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.     

Mutations in both sides of the photosystem I reaction center identify the phylloquinone observed by electron paramagnetic resonance spectroscopy

The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

The mechanism of the degradation of psaB gene product,one of the photosynthetic reaction center subunits of Photosystem I,upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala⁵⁰⁰ and Val⁵⁰¹ on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.     

Assembly of protein subunits within the stromal ridge of photosystem I. Structural changes between unbound and sequentially PS I-bound polypeptides and correlated changes of the magnetic properties of the terminal iron sulfur clusters

The X-ray structure of Photosystem I (PS I) from Synechococcus elongatus was recently solved at 2.5A resolution (PDB entry 1JB0). It provides a structural model for the stromal subunits PsaC, PsaD and PsaE, which comprise the "stromal ridge" of PS I. In a separate set of studies the three-dimensional solution structures of the unbound, recombinant PsaC (PDB entry 1K0T) and PsaE (PDB entries 1PSF, 1QP2 and 1GXI) subunits were solved by NMR. The PsaC subunit of PS I is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters F(A) and F(B), which are the terminal electron acceptors in PS I. Comparison of the PsaC structure in solution with that in the X-ray structure of PS I reveals significant differences between them which are summarized and evaluated here. Changes in the magnetic properties of [4Fe-4S] centers F(A) and F(B) are related to changes in the protein structure of PsaC, and they are further influenced by the presence of PsaD. Based on experimental evidence, three assembly stages are analyzed: PsaC(free), PsaC(only), PsaC(PS I). Unbound, recombinant PsaD, studied by NMR, has only a few elements of secondary structure and no stable three-dimensional structure in solution. When PsaD is bound in PS I, it has a well-defined three-dimensional structure. For PsaE the three-dimensional structure is very similar in solution and in the PS I-bound form, with the exception of two loop regions. We suggest that the changes in the structures of PsaC and PsaD are caused by the sequential formation of multiple networks of contacts between the polypeptides of the stromal ridge and between those polypeptides and the PsaA/PsaB core polypeptides. The three-dimensional structure of the C(2)-symmetric F(X)-binding loops on PsaA and PsaB were also analyzed and found to be significantly different from the binding sites of other proteins that contain interpolypeptide [4Fe-4S] clusters. The aim of this work is to relate contact information to structural changes in the proteins and to propose a model for the assembly of the stromal ridge of PS I based on this analysis.     

Modeling Photosystem I with the alternative reaction center protein PsaB2 in the nitrogen fixing cyanobacterium Nostoc punctiforme

Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster F(X) in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to F(X) and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Consecutive forward electron transfer from A0 TO A1 to FX with anthraquinone in the A1 site as studied by transient EPR

In photosystem I (PS I), phylloquinone (PhQ) acts as a low potential electron acceptor during light-induced electron transfer (ET). The origin of the very low midpoint potential of the quinone is investigated by introducing anthraquinone (AQ) into PS I in the presence and absence of the iron-sulfur clusters. Solvent extraction and reincubation is used to obtain PS I particles containing AQ and the iron-sulfur clusters, whereas incubation of the menB rubA double mutant yields PS I with AQ in the PhQ site but no iron-sulfur clusters. Transient electron paramagnetic resonance spectroscopy is used to investigate the orientation of AQ in the binding site and the ET kinetics. The low temperature spectra suggest that the orientation of AQ in all samples is the same as that of PhQ in native PS I. In PS I containing the iron sulfur clusters, (i) the rate of forward electron transfer from the AQ*- to F(X) is found to be faster than from PhQ*- to F(X), and (ii) the spin polarization patterns provide indirect evidence that the preceding ET step from A0*- to quinone is slower than in the native system. The changes in the kinetics are in accordance with the more negative reduction midpoint potential of AQ. Moreover, a comparison of the spectra in the presence and absence of the iron-sulfur clusters suggests that the midpoint potential of AQ is more negative in the presence of F(X). The electron transfer from the AQ- to F(X) is found to be thermally activated with a lower apparent activation energy than for PhQ in native PS I. The spin polarization patterns show that the triplet character in the initial state of P700)*+AQ*- increases with temperature. This behavior is rationalized in terms of a model involving a distribution of lifetimes/redox potentials for A0 and related competition between charge recombination and forward electron transfer from the radical pair P700*+A0*-.     

Assembly of photosystem I. II. Rubredoxin is required for the in vivo assembly of F(X) in Synechococcus sp. PCC 7002 as shown by optical and EPR spectroscopy

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, F(X), F(A), and F(B), are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubA mutant. The flash-induced decay kinetics of both P700(+) in the visible and A(1)- in the near-UV show that charge recombination occurs between P700(+) and A(1)- in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A(1). In agreement, the spin-polarized X-band EPR spectrum of P700(+) A(1)- at low temperature shows that an electron cycle between A(1)- and P700(+) occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [F(A)/F(B)]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to A(1)- center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of F(X), F(B), and F(A), the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the F(X) iron-sulfur cluster but that F(X) is not required for the biosynthesis of trimeric P700-A(1) cores. Since the PsaC protein requires the presence of F(X) for binding, the absence of F(A) and F(B) may be an indirect result of the absence of F(X).     

A comparative analysis of the spin state distribution of in vitro and in vivo mutants of PsaC. A biochemical argument for the sequence of electron transfer in Photosystem I as FX → FA → FB → ferredoxin/flavodoxin

The EPR properties of in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants of PsaC are compared in an attempt to extract information about electron transfer not contained in any one of these studies in isolation. This analysis indicates that 1) sulfur from an external 'rescue thiolate' is preferred over oxygen from an aspartate or serine replacement amino acid as a ligand to the F_A and F_B iron-sulfur clusters; 2) the inherent spectroscopic symmetry in the F_A and F_B clusters of unbound PsaC is lost when PsaC is docked to its site on the PsaA/PsaB heterodimer; 3) the bound 'rescue thiolate' ligand in the modified site of the F_A cluster, but not the F_B, cluster is displaced when PsaC is docked to its site on the PsaA/PsaB heterodimer; 4) the free energy of binding PsaC to the PsaA/PsaB heterodimer drives the otherwise-unfavorable ligand replacement in the F_A site. These and other findings argue that the substitute ligands support a [4Fe–4S] cluster at the modified site, but the cluster is in either a ground spin state of S 3/2 or S = 1/2 depending on the chemical identity of the ligand, on whether PsaC is unbound or bound, and on the reduction state of the cluster in the unmodified site. By a comparative analysis of the spin state distribution of the in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants, and with knowledge from the X-ray crystal structure that PsaC is bound asymmetrically to the PS I reaction center, an independent case is made that PsaC is oriented so that the F_A cluster is proximal to F_X and the F_B cluster is distal to F_X. These results are compared and contrasted with the results of in vivo mutagenesis studies of PsaC in Anabaena variabilis ATCC 29413 and in Chlamydomonas reinhardtii. In all cases, the primary data can be interpreted to support the sequence of electron transfer as F_X F_A F_B ferredoxin.     

The luminal helix l of PsaB is essential for recognition of plastocyanin or cytochrome c6 and fast electron transfer to photosystem I in Chlamydomonas reinhardtii.

At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c(6) are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated <20% of PSI as compared with wild type and were only able to grow photoautotrophically at low light intensities. Mutant strains E613N, E613K, and W627F accumulated >50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu(613) of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c(6) to PSI. Mutation of Trp(627) to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cyt c(6). It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cyt c(6) and PSI. These data strongly suggest that Trp(627) is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem.     

Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.     

Contributions of the protein environment to the midpoint potentials of the A1 phylloquinones and the Fx iron-sulfur cluster in photosystem I

Electrostatic calculations have predicted that the partial negative charge associated with D575PsaB plays a significant role in modulating the midpoint potentials of the A1A and A1B phylloquinones in photosystem I. To test this prediction, the side chain of residue 575PsaB was changed from negatively charged (D) to neutral (A) and to positively charged (K). D566PsaB, which is located at a considerable distance from either A1A or A1B, and should affect primarily the midpoint potential of FX, was similarly changed. In the 575PsaB variants, the rate of electron transfer from A1A to FX is observed to decrease slightly according to the sequence D/A/K. In the 566PsaB variants, the opposite effect of a slight increase in the rate is observed according to the same sequence D/A/K. These results are consistent with the expectation that changing these residues will shift the midpoint potentials of nearby cofactors to more positive values and that the magnitude of this shift will depend on the distance between the cofactors and the residues being changed. Thus, the midpoint potentials of A1A and A1B should experience a larger shift than will FX in the 575PsaB variants, while FX should experience a larger shift than will either A1A or A1B in the 566PsaB variants. As a result, the driving energy for electron transfer from A1A and A1B to FX will be decreased in the former and increased in the latter. This rationalization of the changes in kinetics is compared with the results of electrostatic calculations. While the altered amino acids shift the midpoint potentials of A1A, A1B, and FX by different amounts, the difference in the shifts between A1A and FX or between A1B and FX is small so that the overall effect on the electron transfer rate between A1A and FX or between A1B and FX is predicted to be small. These conclusions are borne out by experiment.     

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.     

Location of the iron-sulfur clusters FA and FB in photosystem I: an electron paramagnetic resonance study of spin relaxation enhancement of P700+.

Photosystem I (PS I) mediates electron-transfer from plastocyanin to ferredoxin via a photochemically active chlorophyll dimer (P700), a monomeric chlorophyll electron acceptor (A0), a phylloquinone (A1), and three [4Fe-4S] clusters (FX/A/B). The sequence of electron-transfer events between the iron-sulfur cluster, FX, and ferredoxin is presently unclear. Owing to the presence of a 2-fold symmetry in the PsaC protein to which the iron-sulfur clusters F(A) and F(B) are bound, the spatial arrangement of these cofactors with respect to the C2-axis of symmetry in PS I is uncertain as well. An unequivocal determination of the spatial arrangement of the iron-sulfur clusters FA and FB within the protein is necessary to unravel the complete electron-transport chain in PS I. In the present study, we generate EPR signals from charge-separated spin pairs (P700+-FredX/A/B) in PS I and characterize them by progressive microwave power saturation measurements to determine the arrangement of the iron-sulfur clusters FX/A/B relative to P700. The microwave power at half saturation (P1/2) of P700+ is greater when both FA and FB are reduced in untreated PS I than when only FA is reduced in mercury-treated PS I. The experimental P1/2 values are compared to values calculated by using P700-FA/B crystallographic distances and assuming that either FA or FB is closer to P700+. On the basis of this comparison of experimental and theoretical values of spin relaxation enhancement effects on P700+ in P700+ [4Fe-4S]- charge-separated pairs, we find that iron-sulfur cluster FA is in closer proximity to P700 than the FB cluster.