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.

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mtDNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase ␥ (pol ␥). Since Tyr-955 is a highly conserved residue critical for nucleotide recognition among family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (k cat ) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (K m ). The Y955C derivative is 2-fold less accurate for base pair substitutions than wild-type pol ␥ despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10 -100-fold. The error-prone DNA synthesis observed for the Y955C pol ␥ is consistent with the accumulation of mtDNA mutations in patients with PEO.Disruption of mitochondrial energy metabolism causes mitochondrial disorders that play a central role in many degenerative diseases, aging, and cancer. Hundreds of mitochondrial and nuclear gene products are required for the proper functioning of the mitochondria. Accordingly heritable mitochondrial diseases exhibit both maternal and Mendelian modes of inheritance with considerable genetic heterogeneity (1-3).Progressive external ophthalmoplegia (PEO) 1 and mitochondrial neurogastrointestinal encephalomyopathy belong to a subclass of autosomal mitochondrial disorders associated with depletion of the mitochondrial genome and/or the accumulation of mutations and deletions within mtDNA (1, 4 -6). Within the last two years, several nuclear genes controlling maintenance of mtDNA have been identified at disease loci, including the genes for adenine nucleotide translocator 1 (ANT1) at locus 4q34 -35 (7), thymidine phosphorylase at locus 22q13.32-qter (8), a putative mitochondrial helicase (Twinkle) at locus 10q24 (9), an unidentified gene at locus 3p14 -21 (10), and the sole mitochondrial DNA polymerase (pol ␥) at locus 15q22-26 (11). Sequence analysis through the pol ␥ gene (12) in a Belgian pedigree with dominant PEO identified a heterozygous A to G mutation at codon 955 (Y955C) (11). Located in the active site of pol ␥, Tyr-955 is a highly conserved residue among a wide variety of DNA polymerases. As a family A DNA polymerase, pol ␥ is related to Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase, and amino acid sequence alignments reveal that Tyr-955 in pol ␥ is equivalent to Tyr-766 in E. coli pol I and Tyr-530 in T7 DNA polymerase (see Fig. 1A). The three-dimensional structure of T7 DNA polymerase (13) in a ternary complex with DNA and a nuc...

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The prominent basic patch seen in the atomic structure of the lumen-side domain of turnip cytochrome f, consisting of Arg209 and Lys187, 58, 65, and 66, was proposed to be an electrostatically complementary docking site for its physiological electron acceptor, plastocyanin [Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95-105]. This proposal agrees with solution studies on the cytochrome f/plastocyanin electron-transfer reaction that showed a major contribution of electrostatic interactions to the docking, but not with studies on the oxidation rate of cyt f in vivo using mutants in which the basic patch of cyt f was neutralized. The apparent contradiction might be explained by an unknown electron acceptor protein for cyt f. However, (i) flash-induced oxidation of cyt f is absent in a PC-deficient mutant. (ii) Lys58, 65, and 66 in the large domain and Lys188 and 189 in the small domain are major contributors to the ionic strength dependence of the electron-transfer reaction in solution. Replacement of Lys58 and 65 by neutral residues and of Lys66 by the acidic residue Glu66 resulted in a >10-fold decrease in the rate of electron transfer in solution and complete loss of its ionic strength dependence. Replacement of Lys188 and Lys189 in the small domain of cyt f resulted in a 3-4-fold decrease in the second-order rate constant and a smaller dependence of the overall rate of electron transfer on ionic strength, corresponding to a loss of two positive charges. (iii) Acidification of the thylakoid lumen cannot explain the absence of electrostatic interactions. (iv) Changing the five lysines to acidic residues did not result in any significant retardation of the rate of cyt f oxidation in vivo. If the docking of cyt f and plastocyanin in vivo is mediated by basic residues of cyt f, they are different from those that mediate electron transfer in vitro or that are implicated by simulations of electrostatic interactions of the docking. Alternatively, docking of cyt f/PC in vivo is limited by spatial constraints or release of PC from P700 that precludes a rate-limiting mediation of the cyt f/PC reaction by specific electrostatic interactions. The cyt f/PC system in Chlamydomonas reinhardtii is the first electron-transfer couple for which the role of electrostatics in mediating the docking reaction has been studied both in vitro and in vivo.

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.