The prominent interdomain basic surface region seen in the high-resolution structure of the active lumen-side C-terminal fragment of turnip cytochrome f, containing the conserved Lys58,65,66 (large domain) and Lys187 (small domain), has been inferred from in vitro studies to be responsible for docking of its physiological oxidant, plastocyanin. The effect of the putative docking region of cyt f on its reactivity in vivo was tested by site-directed mutagenesis in Chlamydomonas reinhardtii. Three charge-neutralizing mutants were constructed involving: (i)the two lysines (Lys188Asn-Lys189Gln) in the small domain, (ii) the three lysines (Lys58Gln-Lys65Ser-Lys66Glu) in the large domain, and (iii) all five of these lysines spanning both domains. All mutants grew phototrophically. The mutants displayed a 20-30% increase in average generation time, and comparable decreases in rates of steady-state oxygen evolution and the slow (millisecond) electrochromic 515 nm band shift. The magnitude of the changes was greatest in the 5-fold Lys-minus mutant (Lys58Gln-Lys65Ser-Lys66Glu-Lys188Asn-Lys189G ln). The mutants showed a small increase (approximately 25%) in the t1/2, from 0.2 to 0.25 ms, of cyt f photooxidation, far less than anticipated (ca. 100-fold) from in vitro studies of the effect of high ionic strength on the cyt f-PC interaction. The t1/2 of cyt f dark reduction via the Rieske protein increased from 5-6 ms in the wild type to 11-12 ms in the 5-fold Lys-minus mutant. Cells grown phototrophically in the absence of Cu, where cyt c6 is the electron acceptor of cyt f, displayed net rates of cytochrome photooxidation that were slightly faster than those in the presence of Cu, which also decreased by a factor of < or = 25% in the Lys-minus mutants. It was concluded that (a) the net effect of electrostatic interaction between cytochrome f and its electron acceptor in vivo is much smaller than measured in vitro and is not rate-limiting. This may be a consequence of a relatively high ionic strength environment and the small diffusional space available for collision and docking in the internal thylakoid lumen of log phase C. reinhardtii. (b) The efficiency of electron transfer to cytochrome f from the Rieske protein is slightly impaired by the neutralization of the lysine-rich domain.
The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.
The following findings concerning the structure of the cytochrome b 6 f complex and its component polypeptides, cyt b 6 , subunit IV and cytochrome f subunit are discussed: 1.Comparison of the amino acid sequences of 13 and 16 cytochrome b 6 and subunit IV polypeptides, respectively, led to (a) reconsideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cyt b 6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudo-conserved residues) in the segments of cyt b 6 predicted to be extrinsic on the n-side of the membrane. 2.The intramembrane attractive forces between trans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. 3.The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. 4.The isolated cyt b 6 f complex contains one molecule of chlorophyll a. 5.The structure of the 252 residue lumen-side domain of cytochrome f isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 Å.
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