Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc₁ activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc₁ complex.     

Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1 complex of the A1AO ATP synthase

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A₁A_O ATP synthases (A₃B₃CDE₂FG₂ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A₃B₃D- and A₃B₃DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.     

Interaction of the Thermoplasma acidophilum A1A0-ATP synthase peripheral stalk with the catalytic domain

The peripheral stalk of the archaeal ATP synthase (A₁A₀)-ATP synthase is formed by the heterodimeric EH complex and is part of the stator domain, which counteracts the torque of rotational catalysis. Here we used nuclear magnetic resonance spectroscopy to probe the interaction of the C-terminal domain of the EH heterodimer (E_CT1H_CT) with the N-terminal 23 residues of the B subunit (B_NT). The data show a specific interaction of B_NT peptide with 26 residues of the E_CT1H_CT domain, thereby providing a molecular picture of how the peripheral stalk is anchored to the A₃B₃ catalytic domain in A₁A₀.

Structured summary

MINT-7260681: Hct (refseq:NP_393485), Ect1 (uniprotkb:Q9HM68) and Bnt (uniprotkb:Q9HM64) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1-59) of the Saccharomyces cerevisiae V1VO ATPase

Understanding the structural traits of subunit G is essential, as it is needed for V₁V_O assembly and function. Here solution NMR of the recombinant N- (G_1-59) and C-terminal segment (G_61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G_1-59 only. The residues of G_1-59 involved in d binding are Gly7 to Lys34. The structure of G_1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G_1-59 are flexible. The surface charge distribution of G_1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G_1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E_18-38 of subunit E of yeast V-ATPase and the presently solved structure of G_1-59, both proteins have been docked and binding epitopes have been analyzed.     

Synthesis and characterization of carbamoyl methyl pyrazole(CMPz) compounds of palladium(II) chloride: The crystal and molecular structure of [PdCl2{C3H3N2CH2CONBu2}2]

Carbamoyl methyl pyrazole compound of palladium(II) chloride of the type [PdCl₂L₂] (where L =  C₅H₇N₂CH₂CON(C₄H₉)₂, C₅H₇N₂CH₂CON(ⁱC₄H₉)₂, C₃H₃N₂CH₂CON(C₄H₉)₂, or C₃H₃N₂CH₂CON(ⁱC₄H₉)₂) has been synthesized and characterized by IR and ¹H NMR spectroscopy. The structure of the compound [PdCl₂{C₃H₃N₂CH₂CONⁱBu₂}₂] has been determined by single crystal X-ray diffraction and shows that the ligands are bonded through the soft pyrazolyl nitrogen atom to the palladium(II) chloride in a trans disposition.     

Solution-mediated syntheses and characterizations of two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3]

Two new zincophosphites [C₆H₁₄N₂]_0.5[Zn(H₂PO₃)₂] 1 and [C₄H₁₂N₂]_0.5[(CH₃)₂NH₂][Zn₂(HPO₃)₃] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO₄ and HPO₂(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO₄ and HPO₃ units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.     

Synthesis, crystal structure and vibrational spectroscopy of a novel mixed ligands Ni(II) pentaborate: [Ni(C4H10N2)(C2H8N2)2][B5O6(OH)4]2

A novel organic-inorganic hybrid pentaborate [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂][B₅O₆(OH)₄]₂ has been synthesized by hydrothermal reaction and characterized by FT-IR, Raman spectroscopy, elemental analyses and DTA-TGA. Its crystal structure was determined from single crystal X-ray diffraction. The structure consists of isolated polyborate anion [B₅O₆(OH)₄]⁻ and nickel complex cation of [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺, in which the two kinds of ligands come from the decomposition of triethylenetriamine material. The [B₅O₆(OH)₄]⁻ units are connected to one another through hydrogen bonds, forming a three-dimensional framework with large channel along the a and c axes, in which the templating [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺ cations are located. The assignments of the record FT-IR absorption frequencies and Raman shifts were given.     

Short-bite aminobis(phosphonite) containing olefinic functionalities, PhN{P(OC6H3(OMe-o)(C3H5-p))2}2: Synthesis and transition metal complexes

Short-bite aminobis(phosphonite) containing olefinic functionalities, PhN{P(OC₆H₃(OMe-o)(C₃H₅-p))₂}₂ (1) was synthesized by reacting PhN(PCl₂)₂ with eugenol in the presence of triethylamine. The ligand 1 acts as a bidentate chelating ligand toward metal complexes [M(CO)₄(C₅H₁₀NH)₂] forming [M(CO)₄{η²-PhN{P(OC₆H₃(OMe-o)(C₃H₅-p))₂}₂}] (M = Mo, 2; W, 3). The reaction between 1 and [CpFe(CO)₂]₂ leads to the cleavage of one of the P-N bonds due to the metal assisted hydrolysis to give a mononuclear complex [CpFe(CO){P(O)(OC₆H₃(OMe-o)(C₃H₅-p))₂}{PhN(H)(P(OC₆H₃(OMe-o)(C₃H₅-p))₂)}] (4). Treatment of 1 with gold(I) derivative, [AuCl(SMe₂)] resulted in the formation of a dinuclear complex, [(AuCl)₂{PhN{P(OC₆H₃(OMe-o)(C₃H₅-p))₂}₂}] (5) with a Au···Au distance of 3.118(2) Å indicating the possibility of aurophilic interactions. An equimolar reaction between 1 and [Ru(η⁶-p-cymene)Cl₂]₂ afforded a tri-chloro-bridged bimetallic complex [(η⁶-p-cymene)Ru(μ-Cl)₃Ru{PhN(P(OC₆H₃(OMe-o)(C₃H₅-p))₂)₂}Cl] (6). The crystal structures of 1-3 and 5 were established by single crystal X-ray diffraction studies.     

Syntheses and luminescence behavior of tetranuclear copper(I) diynyl complexes: X-ray crystal structure of [Cu4(PPh3)4(μ3-η-CCCCPh)4]

A series of luminescent tetranuclear cuboidal copper(I) diynyl complexes, [Cu₄(PAr₃)₄(μ₃-η¹-CCCCR′)₄] (Ar=Ph, R′=Ph, C₆H₄CH₃-p, C₆H₄OCH₃-p; Ar=C₆H₄CH₃-p, C₆H₄F-p, R′=Ph) has been synthesized and characterized. The X-ray crystal structure of [Cu₄(PPh₃)₄(μ₃-η¹-CCCCPh)₄] has been determined. The origin of the low-energy emission in the complexes is assigned as derived from a metal-centered 3d⁹4s¹ state, mixed with LMCT [diynyl→Cu₄] and IL [π-π*(diynyl)] states.     

Evidence that histidine forms a coordination bond to the A0A and A0B chlorophylls and a second H-bond to the A1A and A1B phylloquinones in M688HPsaA and M668HPsaB variants of Synechocystis sp. PCC 6803

The axial ligands of the acceptor chlorophylls, A_0A and A_0B, in Photosystem I are the Met sulfur atoms of M688_PsaA and M668_PsaB. To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H_PsaA show that there exist low energy conformations with the His coordinated to A_0A and possibly H-bonded to A_1A. Transient EPR studies on M688H_PsaA indicate a more symmetrical electron spin distribution in the A_1A phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150 fs charge separation between P₇₀₀ and A₀ remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A_0A⁻ to A_1A in M688H_PsaA and 48% from A_0B⁻ to A_1B in M668H_PsaB; the remainder recombine with P₇₀₀⁺ with 1/e times of 25 ns and 37 ns, respectively. Those electrons that reach A_1A and A_1B in the branch carrying the mutation are not transferred to F_X, but recombine with P₇₀₀⁺ with 1/e times of ~ 15 μs and ~ 5 μs, respectively. Hence, the His is coordinated to A₀ in all populations, but in a second population, the His may be additionally H-bonded to A₁. Electron transfer from A₀ to A₁ occurs only in the latter, but the higher redox potentials of A₀ and A₁ as a result of the stronger coordination bond to A₀ and the proposed second H-bond to A₁ preclude electron transfer to the Fe/S clusters.     

New aryl phosphinite ligands avoiding ortho-metallation: Synthesis and molecular structures of trans-[PdCl2(PPh2OR)2] and trans-[Rh(CO)Cl(PPh2OR)2] (R = 2,4,6-Me3C6H2; 2,6-Ph2C6H3)

The new aryl phosphinites PPh₂OR (R = 2,4,6-Me₃C₆H₂, 1; R = 2,6-Ph₂C₆H₃, 2) have been prepared from chlorodiphenylphosphine and the corresponding phenols. In these ligands, the ortho-positions of the aromatic phosphite function are blocked by methyl and phenyl substituents, which allows coordination to metal centres without ortho-metallation. Thus, reaction with [PdCl₂(cod)] leads to the complexes trans-[PdCl₂(PPh₂OR)₂] (R = 2,4,6-Me₃C₆H₂, 3; R = 2,6-Ph₂C₆H₃, 4), while the reaction with [Rh₂(CO)₄Cl₂] gives trans-[Rh(CO)Cl(PPh₂OR)₂] (R = 2,4,6-Me₃C₆H₂, 5; R = 2,6-Ph₂C₆H₃, 6). The single-crystal X-ray structure analyses of 3 and 5 confirm the trans-coordination of the new ligands in these square-planar complexes.     

The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine-Binding Specificities

Abstract: Two GABA_A receptor subunit-specific antibodies anti-α₆ and anti-α₁ have been used for elucidating the relationship between the presence of α₁ and/or α₆ subunits in the cerebellar GABA_A receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABA_A receptors have α₆, whereas 76% of the receptors have α₁ as determined by [³H]muscimol binding. Results show that 42–45% of the receptors having α₆ also have α₁, whereas 13–15% of the receptors that contain α₁ also have α₆. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABA_A receptors; i.e., one has both α₁ and α₆ subunits, a second type has α₁ but not α₆, and a third type has α₆ but not α₁ subunits. The results also show that receptors where α₁ and α₆ subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different α subunit. The α₁ subunit contributes the high-affinity binding of [³H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [³H]Ro 15-4513. The α₆ subunit contributes the diazepam-insensitive binding of [³H]Ro 15-4513, but it does not bind [³H]Ro 15-1788 with high affinity. Thus, in the cerebellar α₁–α₆ GABA_A receptors, there is no dominance of the pharmacology of one α subunit over the other.     

Synthesis and characterization of some oxovanadium(V) complexes with internally functionalized oximes: Crystal and molecular structures of heptacoordinated [VO{ONC(CH3)(C4H3O-2)}3] and [VO{ONC(CH3)(C4H3S-2)}3] · 0.5C6H6

New oxovanadium(V) complexes with internally functionalized oximes of the type VO{OPrⁱ}_3−n{ONC(CH₃)(Ar)}_n] (where Ar = C₄H₃O-2, C₄H₃S-2 and C₅H₄N-2 and n = 1-3) have been prepared in quantitative yields by the reaction of VO(OPrⁱ)₃ with the corresponding oximes in various stoichiometric ratios in refluxing anhydrous benzene. The products have been characterized by elemental analyses and spectroscopic (FT IR, ¹H, ¹³C{¹H} and ⁵¹V NMR) studies. FAB mass spectral analysis of [VO{OPrⁱ}{ONC(CH₃)C₄H₃S}₂] indicates the monomeric nature of the complex. ⁵¹V NMR values for these complexes suggest the formation of tetra-coordinate species in solution. However, the single crystal X-ray diffraction studies of [VO{ONC(CH₃)(C₄H₃O-2)}₃] and [VO{ONC(CH₃)(C₄H₃S-2)}₃] · 0.5C₆H₆ exhibit the presence of vanadium(V) atoms in a unique hepta-coordination state with distorted pentagonal bipyramidal geometry in the solid state. The oxo- atom occupies the axial position while the oximato ligands are bonded in a dihapto (η²-N,O) manner with the formation of three membered rings.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

NMR solution structure of the N-terminal domain of subunit E (E<Subscript>1–52</Subscript>) of A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase from <Emphasis Type="Italic">Methanocaldococcus jannaschii</Emphasis>

The N-termini of E and H of A₁A_O ATP synthase have been shown to interact and an NMR structure of N-terminal H_1–47 has been solved recently. In order to understand the E-H assembly and the N-terminal structure of E, the truncated construct E_1–52 of Methanocaldococcus jannaschii A₁A_O ATP synthase was produced, purified and the solution structure of E_1–52 was determined by NMR spectroscopy. The protein is 60.5 Å in length and forms an α helix between the residues 8–48. The molecule is amphipathic with a strip of hydrophobic residues, discussed as a possible helix-helix interaction with neighboring subunit H.     

A novel unexpected luminescent quarternary coordination polymer {Sm3(C8H4O4)4(C12N2H8)2(NO3)}n with three high asymmetrical central Sm fragments by hydrothermal assembly

A novel chain-like luminescent samarium coordination polymer {Sm₃(C₈H₄O₄)₄(C₁₂N₂H₈)₂(NO₃)}_n (C₈H₄O₄ = phthalate, C₁₂N₂H₈ = 1,10-phenanthroline) has been assembled by hydrothermal process. The title complex crystallizes in the monoclinic system, space group P2(1)/c, with lattice parameters a = 22.56(3) Å, b = 11.155(15) Å, c = 20.32(3) Å, β = 96.70(2)°, V = 5078(12) ų, F(000) = 2964, GOF = 0.857, R₁ = 0.0358, wR₂ = 0.0597, Z = 4. Samarium ions exhibit different coordination modes from each other and lead to the unexpected high asymmetrical structure. To our knowledge, it is the first example of lanthanide coordination polymers comprising the three asymmetrical central Sm³⁺ fragments. The photophysical properties have been studied with excitation and emission spectra.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0 reoxidation dynamics in Photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P₇₀₀, and a sequence of electron acceptors, A, A₀ and A₁, bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A₀, are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A₀⁻ (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A₀⁻ on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A₀⁻ is transient, and that reoxidation of A₀⁻ occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A₁. This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A₀ in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.