(Received for publication, January 5, 1996; and in revised form, February 28, 1996)
From the
Ferrochelatase is a mitochondrial inner membrane-bound enzyme
that catalyzes the insertion of ferrous iron into protoporphyrin, the
terminal step in protoheme biosynthesis. The functional/structural
roles of 10 invariant amino acid residues were investigated by
site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast
strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes
were determined for the enzymes associated with the yeast membranes and
the enzymes in the bacterial soluble fraction. They were compared with
the in vivo functioning of the mutant enzymes. The main
conclusions are the following. Glu-314 is critical for catalysis, and
we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding.
Asp-246 and Tyr-248 are also involved in metal binding in a synergistic
manner. The K for protoporphyrin was also
increased in the H235L, D246A, and Y248L mutants, suggesting that the
binding sites of the two substrates are not independent of each other.
The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had
1.2-2-fold increased V
and 4-10-fold
increased K
values for protoporphyrin,
but the amount of heme made in vivo was 10-100% of the
normal value. These mutations probably affected the geometry of the
active center, resulting in improper positioning of protoporphyrin.
Protoheme is an important molecule used to build almost all the cytochromes and hemoproteins in the cells of practically all organisms. A key enzyme in protoheme production is ferrochelatase (EC 4.99.1.1; protoheme ferro-lyase), which catalyzes the insertion of ferrous iron into the tetrapyrrolic nucleus of protoporphyrin IX, at the terminus of the heme biosynthetic pathway. In eukaryotes, ferrochelatase is a peripheral protein associated with the matrix side of the inner mitochondrial membrane. Protoporphyrin is supplied by protoporphyrinogen oxidase, the preceding enzyme in the pathway, also located in the inner mitochondrial membrane. But the nature and location of the iron pool that serves as substrate for ferrochelatase are not known. Yeast and murine ferrochelatases are synthesized as high molecular weight precursors that are proteolytically processed to their mature forms during their import into the mitochondria (see (1, 2, 3, 4) for reviews).
The
ferrochelatases isolated from various sources all have very similar
catalytic properties, indicating that the major features of the
reaction are conserved(1, 2, 3, 4) .
Ferrochelatase genes and cDNAs have now been isolated and sequenced
from bacteria(5, 6, 7) , yeast(8) ,
mammals(9, 10, 11) , and
plants(12, 13) . The deduced amino acid sequences
exhibit 10% identity. The prokaryotic enzymes are
30 amino
acids shorter at the C terminus than the eukaryotic ones. A
[2Fe-2S] cluster was recently found in mammalian
ferrochelatases, probably bound to a cysteine-rich motif in the
carboxyl-terminal extension of the mammalian enzymes, but absent from
the extensions of the yeast and plant
enzymes(14, 15) . The biological role of this cluster
remains to be established.
In spite of many studies, the catalytic mechanism of ferrochelatase is still unknown. Models have been proposed on the basis of kinetic studies, substrate specificity, and chemical modifications of certain amino acid residues and from analysis of the inhibition by N-alkylporphyrins(1, 2, 3, 4, 16) . The active site is believed to be an enclosed hydrophobic pocket(17) , with cysteinyl (18) and arginyl (19) residues implicated in the binding of metal and porphyrin propionate(s). But the fact that no cysteine is conserved in the known ferrochelatase sequences makes it very unlikely that cysteine is involved in metal binding. Whether the binding of the two substrates is random or ordered, the metal binding prior to porphyrin, is not yet clear. Also unclear is the fate of the two protons released from the pyrrolic nitrogen atoms. Distortion of the porphyrin ring has been suggested as a transition-state intermediate facilitating metalation, and aromatic residues might aid in and/or stabilize the bending of a pyrrole ring(1, 16) .
Mutations have been identified that cause amino acid substitutions in human and yeast ferrochelatases, and their effects on the enzyme structure and function have been described(20, 21, 22) . These mutations seem to affect structural aspects of the enzyme, rather than active-site residues specifically involved in the binding of either substrate or in catalysis. Site-directed mutagenesis in human ferrochelatase has recently shown that one strictly conserved histidine residue plays a significant role in metal ion binding(23) . We have now constructed, by directed mutagenesis, single- and double-residue substitutions of 10 invariant amino acids in the yeast ferrochelatase. The mutant enzymes were expressed in a yeast strain from which the ferrochelatase gene HEM15 had been deleted and in Escherichia coli. The functional consequences of these mutations for in vivo heme synthesis were compared with the enzyme defects measured in vitro. The results indicate that the four invariant residues His-235, Asp-246, Tyr-248, and Glu-314 are part of the active center, and their roles in ferrochelatase function are discussed.
Figure 1:
Construction of the pMG1 and pMG2
plasmids. pMG1 was obtained by ligating the 2.5-kb EcoRI-BamHI DNA fragment retrieved from the plasmid
pBluescript SK/HEM15 (8) (after removal of the leftmost BglII site) into plasmid pFL39`, a low copy number TRP1 vector derived from pFL39(25) . pMG2 was obtained by
ligating the polymerase chain reaction-amplified DNA fragment encoding
mature ferrochelatase (hem15p) into the pCASS3 vector (30) , placing ferrochelatase expression under the control of
the E. coli alkaline phosphatase promoter phoA.
Details of these constructions are described under ``Materials and
Methods.'' The dashed lines represent the vectors. The
restriction sites used in this work are indicated: B, BamHI; Bg, BglII; Bs, BstEII; E, EcoRI; N, NdeI; S, SalI; St, StuI.
The pMG2 plasmids carrying the
wild-type and mutant ferrochelatases were transformed into E. coli strain DH5 (Life Technologies, Inc.). The bacteria were grown
to saturation in LB/ampicillin medium, diluted 1:100 into complete low
phosphate/MOPS medium containing 100 mg/liter ampicillin(29) ,
and incubated for 18-20 h at 37 °C. The cells were harvested
by centrifugation, resuspended in 0.1 M Tris-HCl buffer (pH
7.6) containing 20% glycerol and 10 µg/ml phenylmethylsulfonyl
fluoride, and disrupted by sonication. The lysed cells were centrifuged
(100,000
g, 1 h, 4 °C) to separate the membrane
and soluble fractions. The membrane pellets were resuspended in the
above buffer. All fractions were analyzed immediately or stored at
-20 °C.
Yeast mitochondrial membranes were prepared as
described(20) . Ferrochelatase activity was monitored
spectrofluorometrically by directly recording the rate of
zinc-protoporphyrin formation as described previously in
detail(3, 20, 31) . The reaction mixture
(3-ml final volume) contained 0.1 M Tris-HCl (pH 7.6), 0.2
mg/ml Tween 80, 0.03-15 µM protoporphyrin (in 0.1 M Tris-HCl (pH 7.6), 1% Tween 80), and 0.2-25 µM zinc (ZnSO5H
O dissolved in water);
the reaction was initiated by adding the enzyme. The activity was
expressed as nmol of zinc-protoporphyrin/h/mg of protein. The kinetic
data were analyzed graphically and using the random Bi Bi fit of the
EZ-FIT curve-fitting program(33) .
Figure 2: Amino acid sequence of yeast ferrochelatase showing location of residues mutated in this study. The presequence is underlined, and the cleavage site of the precursor form is indicated by an arrow. Invariant residues among ferrochelatases from diverse species are marked by asterisks(3) . Amino acids substituted in this study are boxed. Overlined amino acids mark positions where substitutions have been identified in hem15 mutants(20, 22) and in human ferrochelatase(21) .
Figure 3:
Growth phenotypes associated with the
expression of the mutant ferrochelatases. The yeast strain AS1 carrying
the null allele hem15(-) was transformed with
plasmid pMG1 expressing wild-type (WT) or mutant forms of
ferrochelatase. The AS1 strain and the different transformants were
grown in YPGTe. Drops (3 µl) of each culture were spotted onto
different media in 10-fold serial dilutions, and the plates were
incubated at 30 °C for 2 days (YPG supplemented or not) or 6 days
(YPGly). YP plates contained 2% glucose (YPG) or 2% glycerol (YPGly),
supplemented with heme (YPGheme) or Tween 80 + ergosterol
(YPGTe).
There was a good inverse relationship between the amounts of hemes and those of total porphyrins (intracellular + excreted in the medium) synthesized by the mutant ferrochelatases in vivo: the less hemes, the more porphyrins, mainly protoporphyrin (>90%). However, two mutants departed from this, R87A and Y95L, which accumulated porphyrins in spite of almost normal heme synthesis.
Figure 4: Immunodetection of mutant ferrochelatases in S. cerevisiae membrane and E. coli soluble fractions. Yeast membrane proteins (25 µg) (row Y) and E. coli soluble proteins (0.3 µg) (row E) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were reacted first with yeast ferrochelatase antiserum and then with alkaline phosphatase-conjugated anti-rabbit secondary antibodies. Yeast and bacterial samples were from the same preparation used to measure ferrochelatase activity. Lane 2, control samples expressing no ferrochelatase (yeast strain AS1, E. coli carrying pCASS3); lane 15, yeast membrane proteins (25 µg) containing wild-type (WT) ferrochelatase. Gels were run for a long time to show the difference in the electrophoretic mobility of the F308A mutant protein.
The H235L, D246V/Y248F, and E314A mutant
ferrochelatases had barely detectable activities. Even when measured
with high substrate concentrations (15 µM protoporphyrin,
50 µM Zn), the activity was <1% of
the wild-type value. The kinetic parameters could not be determined
because of this very low activity. The D246A/Y248L mutant (which
carried less conservative amino acid substitutions than the D246V/Y248F
mutant) had 13% of the wild-type activity, but the K
for both substrates was increased
40-fold. All the other
mutant enzymes, including the single mutants D246A and Y248L, had
higher activities than the wild-type enzyme and higher K
values for protoporphyrin (4-12-fold the normal value). The K
for Zn
of these mutant enzymes
could not be measured precisely because they were already saturated
with the endogenous Zn
present in the assay (reaction
mixture + membranes), as was the wild-type ferrochelatase.
Therefore, the K
for Zn
of these
enzymes was considered to be similar to that of the wild-type enzyme (a
2-fold increase in the K
for Zn
could have been estimated if this had been the case).
The kinetic
parameters of the wild-type and mutant ferrochelatase activities
measured in the bacterial soluble fractions are reported in Table 2. As for the yeast membrane-bound enzymes, reliable
measurements of K for Zn
were
not possible for the wild-type and some mutant enzymes because they
were almost saturated with endogenous Zn
. However, we
estimated that they were close to 0.2 µM, the value of the
purified ferrochelatase(31) . The two H235L and D246V/Y248F
mutant ferrochelatases had K
values for
Zn
that were dramatically increased compared with
that of the wild-type enzyme; their K
values for
protoporphyrin were also greatly increased, and their V
values were 25 and 2% of the wild-type value, respectively. The
D246A single substitution caused an increase in the K
values for both substrates, while the Y248L single substitution
affected only the affinity for protoporphyrin; they did not much affect
the maximal velocity. In contrast, introducing these two mutations
together caused a >90% decrease in the activity, without further
affecting the K
value for either substrate.
Interestingly, the E314A mutant had K
values
identical to those of the wild-type enzyme, although the mutant
displayed only <2% of the wild-type activity. The three other
mutations, Q273E, W282L, and F308A, caused 8-23-fold increases in
the K
for protoporphyrin without impairing the
activity much.
Palmitic acid has been reported to be required for
full activity of the purified yeast ferrochelatase(31) .
Therefore, we tested to see if it had any effect on the enzyme
overexpressed in a soluble form in E. coli. Addition of 1
mM palmitic acid (20 µl/assay of 20 mg of palmitic acid/ml
of dimethyl sulfoxide) did not change the maximal velocity, but
decreased the K for protoporphyrin (0.06
µM) of the wild-type enzyme 3-4-fold. Mutants H235L,
D246A, F308A, and E314A and the double mutants were totally inhibited,
in a dose-dependent manner, by 1 mM palmitic acid. The
activities of mutants Y248L and Q273E were increased 1.5-2-fold,
and their K
for protoporphyrin decreased
1.5-3-fold. Palmitic acid had no effect on mutant W282L. Similar
results were obtained with the mutant enzymes expressed in yeast in a
membrane-bound form. 1 mM palmitic acid inhibited mutants
D246A, D246A/Y248L, and F308A, while it increased the V
of mutants R87A, Y95L, Q111E, Y248L, and Q273E 2-3-fold
without greatly affecting their K
for
protoporphyrin (a 2-fold increase at the most). The role of fatty acids
in the activity of ferrochelatase is
unclear(1, 2, 3, 4) . Our results
favor the idea that they might interfere with the delivery of exogenous
substrates to the active site, rather than promoting the proper active
conformation of the enzyme.
This study describes the replacement by site-directed mutagenesis of 10 invariant amino acids in the yeast ferrochelatase. The function of the mutant enzymes was analyzed in vivo by expressing them in a yeast ferrochelatase-deficient strain and in vitro both in their physiological environment when bound to yeast mitochondrial membranes and as soluble mature-sized proteins overexpressed in E. coli. There was in general a good correlation between the results obtained with these three approaches despite some variations that are discussed below with the analysis of each mutation.
The F308A mutant ferrochelatase had a puzzling phenotype. Its capacity to make heme in vivo was considerably decreased (90%), while it had a normal zinc chelatase activity measured in vitro, albeit with slightly (2.5-4-fold) lower affinity for protoporphyrin. The protein migrated a little slower on electrophoresis than the wild-type and all other mutant ferrochelatases. One possible explanation is that the wild-type enzyme retained some local residual microstructure stabilized by Phe-308 that prevented complete unfolding. The changing of Phe-308 to Ala would destroy this microstructure, promoting full denaturation of the protein, which would then migrate more slowly. This structure may be important in vivo for the proper access of protoporphyrin to the active site when this substrate is generated enzymatically within the mitochondrial membrane or for the correct and efficient release of heme probably also in the membrane.
The D246A mutant ferrochelatase was 5-fold less
active than normal in vivo, and the in vitro analysis
suggested that this was due to a 12-fold lower affinity for
protoporphyrin. However, when expressed in E. coli, the mutant
enzyme displayed increased K
values for both
protoporphyrin (21-fold) and metal (14-fold). The reason(s) for this
discrepancy is not clear at present. The replacement of the neighboring
Tyr-248 with Leu was benign: the function of the mutant enzyme was
normal in vivo, and its catalytic efficiency (V
/K
) measured in vitro was decreased (2-3-fold) only for protoporphyrin. Therefore,
both of the mutations D246A and Y248L seemed to affect mainly the
affinity of ferrochelatase for protoporphyrin. But introducing the two
mutations together was highly detrimental to ferrochelatase function
both in vivo and in vitro. The catalytic efficiency
of the D246A/Y248L double mutant enzyme was now considerably reduced
for both substrates: 300-fold for protoporphyrin and 250-fold for zinc
for the enzyme expressed in yeast; similar values were obtained for the
enzyme expressed in E. coli. The defects of the enzyme were
still aggravated when Asp-246 and Tyr-248 were replaced by Val and Phe,
respectively, although these substitutions should introduce less steric
constraint on the protein (the D246A/Y248L mutant protein was less
stable in E. coli grown at 37 °C compared with
D246V/Y248F). Thus, it appears that it is the combined effects of the
two mutations that caused a decrease in the activity of ferrochelatase
and in its affinity for both substrates. The basis for this synergistic
action is not understood.
The finding that His-235, Asp-246, and Tyr-248, which are clustered in the same region, serve as metal ligands is in complete agreement with the results of Franco et al.(37) , who used Mössbauer spectroscopy to characterize the ferrous ion-binding site. The data indicated an ionic coordination environment for the high spin ferrous ions, consisting of nitrogen- and/or oxygen-containing ligands. Other residues, i.e. the invariant residues Ser-233 and Ser-167, could also be part of the metal-binding site.
The fact that the K values for both metal and protoporphyrin were
increased in these mutants is not unprecedented. The mutations S169F,
S174P, and L62F also caused an increase in the K
values for both substrates, together with an increase in V
(20, 22) . We suggested that the
binding sites of the two substrates are not independent of each other.
One possibility is that one (or both) flexible propionate side chain of
the porphyrin is hydrogen-bonded to some residues of the metal-binding
site. Altering one of these residues or the geometry of the active site
might destabilize the propionate hydrogen-bonding network and weaken
the porphyrin-enzyme interactions. It is conceivable that the
propionate carboxylic group(s) might play a role in the metal binding
process.
In conclusion, this initial mutational survey of the active-site amino acid residues in the yeast ferrochelatase has answered some questions about the residues implicated in substrate binding and catalysis. Further detailed kinetic and inhibition studies with purified mutant enzymes are needed before a more refined model of the active center can be proposed. However, understanding the catalytic mechanism of ferrochelatase will have to wait for the elucidation of its three-dimensional structure. Fortunately, this is in sight since a preliminary x-ray analysis of B. subtilis ferrochelatase crystals was recently reported(39) .