(Received for publication, November 16, 1995; and in revised form, January 16, 1996)
From the
Mitochondrial encoded subunit II of cytochrome c oxidase carries the metal center, which acts as the initial acceptor of electrons from cytochrome c. Among the conserved features of this protein is a region in which five aromatic and three non-aromatic amino acids are conserved in a wide variety of organisms. This aromatic region has been postulated to be involved in transfer of electrons from the copper center in subunit II to the remaining metal centers of cytochrome oxidase in subunit I. To test the functional importance of two conserved, aromatic tryptophan residues and one conserved, non-aromatic glycine residue, yeast strains with alterations at these positions were characterized. The strains with altered codons were tested for their ability to carry out cellular respiration, for their growth rates on non-fermentable carbon sources, and for their cytochrome c oxidase activity. The results demonstrate that the aromatic character of the tryptophan residues appears necessary for subunit II function, while the conserved glycine can be replaced with other, small, uncharged residues.
Cytochrome c oxidase is the terminal protein complex of the electron transport chain, responsible for transferring electrons from cytochrome c to molecular oxygen. In eukaryotes, the three largest subunits, subunits I, II, and III, are encoded on mtDNA, while the remaining subunits are encoded on nuclear DNA(1, 2) . Aerobic bacteria have only three subunits in a completely functional cytochrome oxidase complex. These prokaryotic subunits are homologous to eukaryotic subunits I, II, and III(3) . It therefore seems likely that subunits I, II, and III of the eukaryotic complex comprise the catalytic core of the enzyme, while the remaining nuclear encoded subunits may be involved in regulation or the assembly of the complex.
Deletion of the subunit
III gene from the bacterium Paracoccus denitrificans by in
vitro mutagenesis causes disruption in the assembly of the oxidase
complex, but removal after assembly does not appear to abolish electron
transport and proton pumping activity (4) . These results
suggest that subunits I and II are directly responsible for the
catalytic activity of the enzyme. In addition, the five metal centers
associated with the cytochrome c oxidase complex are bound
exclusively by subunits I and II. Heme a, heme a, and Cu
are associated with subunit
I, and subunit II binds a binuclear metal center,
Cu
-Cu
, which is the site of the initial redox
reaction with cytochrome c(5, 6, 7) . A better understanding
of the mechanism of cytochrome c oxidase function may
therefore be achieved by focusing on subunits I and II.
Subunit II is of interest because it is the primary acceptor of electrons from cytochrome c(8, 9) . Subunit II contains several regions of conserved amino acids from organisms as diverse as prokaryotes, fungi, plants, and humans, including two conserved stretches of hydrophobic amino acids, presumed to span the inner mitochondrial membrane, a number of amino acids that act to bind the binuclear copper center, and a highly conserved aromatic region(10) . The aromatic region is a sequence of 10 consecutive amino acids, including five aromatic and three non-aromatic which may mediate electron transfer from the binuclear copper center in subunit II to heme a in subunit I(10) . To determine if a number of these conserved residues are essential for protein function, and in particular, if the aromatic character of two of the conserved amino acids is important, point mutations in the subunit II gene of Saccharomyces cerevisiae that alter two highly conserved aromatic residues and one conserved non-aromatic residue were analyzed. The consequences of these amino acid substitutions with respect to respiratory competence, growth rates, and cytochrome c oxidase activity are reported.
The entire cox2 gene was amplified and sequenced in each of these mutant strains. In m5301, codon 121 has been altered from GGA (glycine) to GAA (glutamic acid), while in VC20, codon 126 has been changed from TGA (tryptophan) to TGT (cysteine). VC21 carries two mutations, one that converts a non-conserved TAT (tyrosine) codon at position 59 to AAT (asparagine) and one that converts codon 124 TGA (tryptophan) to AGA (arginine). The mutation at codon 59 is likely to be irrelevant since this position is not conserved and since our reversion analysis (see below) demonstrates that recovery of respiratory function depends only on the TGA 124 codon. As shown in Fig. 1, the three mutations of interest are located in the conserved aromatic region of the gene. To determine if each of these residues is absolutely essential for activity and if the aromatic character of the two tryptophan residues is important for their function, derivatives of each of the mutants which had spontaneously recovered respiratory function were isolated and the alterations in these revertant strains were analyzed.
Figure 1: Schematic diagram of the COXII protein. The aromatic region of the protein has been expanded to show the position of the mutations in the strains analyzed. Conserved amino acids are marked with stars.
Since the mutant pet9 allele also causes respiration
deficiency, the presence of this mutation requires that
respiration-proficient revertants be isolated from a diploid
heterozygous for the pet9 mutation. Consequently, each of the
mutant strains was crossed to a strain to give a
diploid of the following genotype (mata/
, +/ade1, +/his4, +/pet9 (cox2)). Recovery of respiration in such a diploid
requires reversion only of the cox2 mutation. For each mutant
strain, isolated diploid colonies were grown overnight in
glucose-containing medium, and the cells spread on two agar plates
containing glycerol and ethanol. The three respiration-deficient
diploids are incapable of growing on this medium, and therefore any
colonies that arise from these diploid cells must carry a dominant
suppressor of the original mutation. The pairs of plates were incubated
at 28 °C and 18 °C and examined periodically over a 4-week
period. Colonies that arose were colony-purified and subjected to
further analysis. To ensure that the analysis did not include cells
derived from a single mutational event, only one colony arising on a
plate on a particular day was chosen for further analysis, unless the
two colonies differed significantly in size. Since colony size is a
reflection of growth rate, colonies of different size are unlikely to
be derived from sibling cells. Furthermore, colonies that arose on
different days are also unlikely to be siblings. This conjecture is
based on evidence that early arising and late arising mutations, seen
under selective conditions in both Escherichia coli and S.
cerevisiae, are not due to the same mutational mechanisms and
exhibit a spectrum of nucleotide changes(18, 19) .
Therefore, colonies arising on different days that were derived from
the same original culture were retained for further analysis.
The
frequency with which revertants appeared was variable in the three
mutant strains. From 60 5-ml cultures of m5301D, 26 revertants were
identified (1.4 10
). Twelve revertants were
identified from 40 5-ml cultures of VC21D (1
10
), while a very large number of revertants (more
than 100) appeared on plates derived from 40 5-ml cultures of VC20D (1
10
). 51 of the latter were ultimately
characterized by sequencing.
Fourteen of the revertants retain the mutant cysteine codon. Since this codon leads to respiration deficiency in the original mutant strain, the sequence of the remainder of the COX2 gene was determined for two of the cysteine revertants. For both of these, the remainder of the gene is wild type, indicating that the alteration responsible for recovery of function resides outside the COX2 locus.
To test
this hypothesis, the original wild type parent, JM22, was crossed to a
strain to create a wild type diploid parent, isogenic
to the diploid revertants. The diploid parent, JM22D, and a number of
VC20 diploid revertant strains were plated under non-selective
conditions, on YPD plates, to obtain single colonies. These colonies
were then replica-plated to YEPG plates and grown at 28 °C to
assess the stability of the respiration-proficient phenotype. As shown
in Fig. 2, for the wild type parent and two other VC20
revertants, one carrying a phenylalanine codon and one carrying a
tyrosine codon, essentially all the colonies grow on YEPG. In contrast,
for two independent cysteine codon carrying mutants, essentially all
the colonies fail to grow on YEPG. Clearly the respiration-proficient
phenotype of these revertants can be maintained only under selective
pressure.
Figure 2: Stability of the respiration-proficient phenotype in isogenic wild type and revertant strains of VC20 (W126C). Cells were grown on non-selective medium (YPD), then replica-plated to selective medium (YEPG) and the plates incubated for 2 days.
The most likely explanation for all spores being
respiration-deficient is that the diploid revertant strain is
heteroplasmic, containing two different mitochondrial genomes. One of
these genomes would be the original mitochondrial DNA, bearing the VC20
mutation, while the other would be a genome
from which most of the mitochondrial DNA has been lost, but on which
the compensatory change resides. While both are maintained under
selective pressure, the heteroplasmic condition is unstable; therefore,
when the cells are grown in glucose, i.e. under non-selective
conditions, one or the other of the mitochondrial genomes is rapidly
lost, resulting in cells that are respiration-deficient(23) .
Since the two mitochondrial genomes segregate from one another at high
frequency, it should be possible to identify spores that carry only the
suppressor genome and not the genome bearing the VC20 mutation.
Identification of such a spore is under way to characterize the nature
of the suppressor mutation.
Figure 3: Effect of substitutions at codon 121. Isogenic diploid strains were grown on non-fermentable carbon sources at 18, 28, and 37 °C.
When a similar growth experiment was carried out for revertants bearing substitutions at codon 126, diploid revertants bearing either a wild type tryptophan codon or a phenylalanine codon grow comparably to wild type, while cysteine and tyrosine codon carrying revertants grow more slowly. As shown in Fig. 4, two independent cysteine codon revertants grow at different rates, suggesting that one of these strains might have incurred a second mutation which also affects the ability to grow on non-fermentable carbon sources, or that the nature of the suppressor in these two strains is different.
Figure 4: Effect of substitutions at codon 126. Isogenic diploid strains were grown on non-fermentable carbon sources at 18, 28, and 37 °C.
Since growth on agar plates is a crude measure of respiratory ability, mitochondria were isolated from diploid strains bearing amino acid substitutions and cytochrome c oxidase activity was measured. As shown in Table 4, replacement of glycine at codon 121 by alanine or serine appears to have little effect on cytochrome oxidase activity. At codon 126, strains bearing TGA and TGG tryptophan codons and TTT phenylalanine codons have wild type levels of cytochrome oxidase activity. On the other hand, replacement with a TAT tyrosine codon leads to an 80% reduction in activity. Two different cysteine codon carrying strains were analyzed. One of these, MODR30D has about 18.5% of the cytochrome oxidase activity seen in wild type while the other, TTDR4, has only 2.8% of wild type activity. These results are consistent with the growth rates determined on YEPG plates, where TTDR4 (cysteine) grew most slowly and MODR30D (cysteine) and MODR29B (tyrosine) grew at intermediate rates.
The three mutant strains characterized bear alterations in conserved amino acids found in the aromatic region of the subunit II protein, which has been suggested to play a role in electron transfer (10) . However, reversion analysis suggests that some amino acid substitutions at these three sites are permissible.
Fourteen revertants were isolated
that still carry the mutant cysteine codon. The complete sequence of
subunit II has been determined for two independent cysteine revertants.
In both cases the remainder of the gene is wild type. Therefore, a
compensatory change must have arisen outside the COX2 locus.
For five of the cysteine codon revertants, the respiration-proficient
phenotype is highly unstable and yields almost all
respiration-deficient spores. These results are consistent with the
diploid revertants being heteroplasmic, bearing both the genome
carrying the VC20 mutation and another, genome,
which bears a suppressor of this mutation. Either genome alone leads to
respiration deficiency, since one bears the cox2 mutation,
while the other carries the suppressor but is missing other
mitochondrial genes that are required for respiration. It is not yet
known if all the cysteine codon revertants are suppressed by a similar
mechanism. At least one of the revertants, TTDR4, has a very slow
growth rate relative to other cysteine codon revertants, but slow
growth may be due to the presence of another unrelated mutation that
also affects respiratory function. One possible candidate for the
suppressor mutation is one of the tRNA
genes. Two
mitochondrial genes for tRNA
, bearing the same anticodon
have been reported(24) , so mutation of one of these to
recognize the cysteine codon but to insert an aromatic amino acid is a
potential mechanism of suppression. Characterization of these
suppressors is under way.
In summary, the analysis has demonstrated that conserved amino acids in the aromatic region of subunit II are important for its function. Reversion analysis suggests that the conserved glycine at position 121 may be important for structural reasons, since the only substitutions detected were with amino acids similar in size and nature. In addition, the aromatic character of the tryptophans at positions 124 and 126 is important for the function of the protein, and they cannot be replaced by non-aromatic amino acids. Tryptophan 126 can be replaced, without loss of function by phenylalanine, and replacement by tyrosine results in reduction but not elimination of cytochrome c oxidase activity. These results are consistent with the hypothesis that the aromatic region functions in electron transfer.