THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 12, Issue of April 25, pp.

8541-8546, 1993
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U. S.A.

Structure-Function Studiesof Yeast Ferrochelatase

IDENTIFICATIONANDFUNCTIONALANALYSISOFAMINO ACID SUBSTITUTIONSTHATINCREASE
V,,, AND T H E KM FORBOTHSUBSTRATES*

(Received for publication, October 5 , 1992)

Abdelhamid Abbasand Rosine Labbe-Bois$

From the Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Uniuersite Paris 7, 2 Place Jussieu, 75251 Paris
Cedex 05, France

The molecular basis of the ferrochelatase defects was together. The hydropathy profile reveals no apparent mem-
investigated intwo “protoporphyric” and partially brane-spanning segment, indicating that ferrochelatase is pe-
heme-deficient yeast mutants. Ferrochelatase, a mito- ripheral.
chondrial inner membrane-bound enzyme, catalyzes A model has been proposed for the mechanism of action of
the incorporation of ferrous iron into protoporphyrin, ferrochelatase, on the basisof kinetic studies, chemical mod-
the last step in protoheme biosynthesis. The mutant ifications of sulfhydryl and arginyl residues, and from the
cells made normal amounts of normal-sized ferroche- analysis of thestronginhibition by N-alkylporphyrins
latase, as detected by immunoblotting. The mutations (Dailey, 1990 and references therein). This model implicates
were identified by sequencing the mutant hem15 al-
cysteine and arginine residues in the binding of metal and
leles amplified in vitro from mutant strains genomic
DNA. A single nucleotide change, causing an amino porphyrin propionate(s),respectively, and involves distortion
acid substitution, was found in each mutant. Substitu- of the porphyrin ring as a transition-state intermediate. It
tion of the conserved Ser-169 by Phe caused a 10-fold has recently received strong support with the finding that
increase in V,, and a 45- and 35-fold increase in the antibodies elicited to a distorted N-methylporphyrin (transi-
KM for protoporphyrin and metal, respectively. Re- tion-state analog) could catalyze metal ion chelation by the
placement of Ser-174 by Pro produced the same ef- planar porphyrin (Cochran and Schultz, 1990). But the fact
fects, but to a lesser degree. There was a good corre- that no cysteine is conserved in the known ferrochelatase
lation between the ferrochelatase defects measured in sequences makes itunlikely that cysteine(s)may form part of
vitro and the heme synthesis deficienciesestimated in the binding sitefor metal substrate.
vivo. The decreased in vivoheme synthesis is probably No mutational analysis of ferrochelatase has yet been re-
due to the lower affinity of the mutant enzymes for ported which could help investigate the structural and func-
iron. We propose that the region identified by the two tional requirements of the enzyme. Ferrochelatase activity is
close mutations contributes to the binding domains of decreased in the human andbovine hereditary disease, eryth-
metal and protoporphyrin. ropoietic protoporphyria, in which excessive protoporphyrin
accumulates in various tissues, causing cutaneous photosen-
sitivity (Nordmann and Deybach, 1990). Some enzymic de-
The chelation of ferrous iron by the tetrapyrrolic macro- fects have been identified in protoporphyria, but the muta-
cycle protoporphyrin IX to form protoheme is the final step tional events responsible for them are unknown (Bloomer et
in the heme biosynthetic pathway and is catalyzed by the al., 1987; Straka et al., 1991; Blom et al., 1990). On the other
membrane-boundenzymeferrochelatase(protohemeferro- hand, a number of mutations causing amino acid substitutions
lyase EC 4.99.1.1). In eukaryotic cells, where it has been most have been reported in human (Lamoril et al., 1991) and E.
studied, theenzyme is associated with the inner mitochondrial coli (Miyamoto et al., 1991; Nakahigashi et al., 1991) ferroche-
membrane, with the active site facing the matrix compart- latases, but the nature of the enzyme dysfunction was not
ment, (Dailey, 1990, and references therein). The ferrochela- analyzed.
tases isolated from rat (Taketani and Tokunaga, 1981) and The present work describes two amino acid substitutions
bovine (Taketani and Tokunaga, 1982)liver and from the in theS. cerevisiae ferrochelatase which lead to increased K M
yeast Saccharomyces cereuisiae (Camadro and Labbe, 1988) for both metal and protoporphyrin and to an increasedVmax.
have very similar physicochemical and catalytic properties. We have also analyzed the phenotypicconsequences of these
Ferrochelatase cDNAs or genes have been isolated and se- enzymic defects. The implicationof the mutatedregion in the
quencedfrommouse(Taketani et al., 1990; Brennerand binding of substrates at the active site of ferrochelatase is
Frasier, 1991), human (Nakahashi et al., 1990), S. cerevkiae discussed.
(Labbe-Bois, 1990), Escherichia coli (Miyamoto et al., 1991)
and Bradyrhizobium japonicum (Frustaci andO’Brian, 1992). MATERIALS ANDMETHODS
The derived amino acidsequences show from 22 to 88% Strains, Media, and Cultivation-The S. cereuisiae mutant strains
identity when analyzed in pairs and10% identity when taken Sm12 (Mata leul arg4 cttl-1 hem15-3) and Sm41 (Mata leul arg4
ctal-1 hem15-4) were isolated from the parental strains DCT1-3D
* This work was funded by the Centre National de la Recherche and DCT3-4A, respectively (Kurlandzka and Rytka, 1985). Since the
Scientifique and by the Universitb Paris 7. The costs of publication strain DCT3-4A no longer exists, the strain DCT1-3D was used as
of this article were defrayed in part by the payment of page charges. reference wild-type HEM15 strain for both mutant strains. The cells
This article must therefore be hereby marked “aduertisement” in were grown at 30 “C with vigorous agitation and aeration in complete
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. medium (1%yeast extract, 1%bactopeptone) with either 2% glucose
$ T o whom correspondence should be addressed: Tel.: 33- (YPG) or 2% ethanol + 2% glycerol (YPE) as carbon source. The
143540479;Fax: 33-144275716. medium was occasionally supplemented with 0.2% Tween 80 (YPGT,

854 1

This is an Open Access article under the CC BY license.

a542 Yeast Ferrochelatase Mutations
YPET). Iron-rich medium was supplemented with 0.18 mM FeCb + iron was quantified spectrophotometrically after its chelation with
4 mM sodium citrate.
E. coli strain DH5a (Bethesda Research Laboratories) was used as
the bacterial host for plasmid cloning and proliferation.
Isolation and Sequencing of the hem15 Mutant Alleles-Genomic
DNA wasprepared from the parental and mutant strains describedas
(Sherman etal., 1986).It was used as template (1fig) foramplification
of the HEM15 alleles by the polymerase chain reaction employing
the GeneAmp kit (Perkin-Elmer Cetus). Two oligonucleotides, cor-
responding to nucleotides -54 to -34 of the coding strand (relative
tothe A + l of the initiator ATG), 5"CCGGAATTCACACA-
TACCTGCTATTTGGAC, and tonucleotides 1207-1187 of the non-
coding strand, 5'-CCCAAGCTTTGAGATTGTGGGATGAATGG,
were synthesized with the addition of an EcoRI or a Hind111 site
(underlined) to the 5' end, respectively. Amplification was carried
out with 30 cycles of 1 min denaturation at 90 "C, 1 min annealing at
55 "C, and 3 min extension at 70 "C. The resulting amplified DNA
fragments (1.26 kilobases) were cloned into pBluescript (Stratagene).
Plasmid DNA isolated from 10 individual transformants for each
strain was combined to alleviate possible errors introduced by the
Tacq polymerase and sequenced using the Sequenase polymerase (U.
S. Biochemicals), [ w ~ ~ S ] ~ A(Amersham),
TP and HEM15-specific
oligonucleotideprimers.
Ferrochelatase Immunodetection and Activity Assay-Total pro-
teins extracted from trichloroacetic acid-treated cells and proteins
from the membrane fraction in 0.05 M NaOH were resolved by SDS-
polyacrylamide gel electrophoresis and electrophoretically transferred
to nitrocellulose membranes. An alkaline phosphatase-coupled sec-
ondary antibody (Promega) was used to visualize the anti-yeast
ferrochelatase antibody prepared by J. M. Camadro (Camadro and
Lahbe, 1988).
Membrane-bound ferrochelatase was assayed spectrofluorometri-
cally by measuring the rate of zinc-protoporphyrin formation (Ca-
madro and Labbe, 1988). Mitochondrial membranes were prepared
as follows. Cells were harvested in the mid-log phase of growth in
YPET medium, washed with water, suspended in 0.1 M Tris-HC1, pH
7.6, and mechanically disrupted with glass beads. The resulting cell
homogenate was cleared of debris by centrifugation at 5,000 X g for
5 min. The membrane fraction was pelleted a t 100,000 X g for 60
min. Membranes were washed once with 0.1 M Tris-HC1, pH 7.6,
resuspended a t a protein concentration of 20-30 mg/ml, and stored
at -70 "C. Proteins were determined by the method of Lowry using
bovine serum albumin as standard.
Ferrochelatase activity was monitored by directly recording the
rate of zinc-protoporphyrin formation at 30 "C with a Jobin-Yvon
JY3-D spectrofluorometer equipped with a thermostated cell-holder
allowing magnetic stirring in the cuvette and a red-sensitive Hama-
matsu R928H4 photomultiplier tube. The maximum excitation and
emission wavelengths for zinc-protoporphyrin under the assay con-
ditions were 418 and 588 nm, respectively. The reaction mixture (3
ml final) contained 0.1 M Tris-HC1, pH 7.6, 0.2 mg/ml Tween 80
(including the amount of Tween 80 added with the protoporphyrin
solution), protoporphyrin, and zinc. The reaction was initiated by
adding 0.1-0.5mgof membrane protein. Stock solutions of 1 mM
protoporphyrin IX disodium salt (Serva) in 0.1 M Tris-HC1, pH 7.6,
containing 1%(w/v) Tween 80 were diluted with the same buffer just
before use; their absorption spectra were run to check for monomer-
ization of protoporphyrin. Zinc wasprepared as a 3mM stock solution
of ZnSOl. 5H20in water. Pure zinc-protoporphyrin (Porphyrin Prod-
ucts, Logan, UT) was used to calibrate the assay. Ferrochelatase
activity was expressed as nanomoles of zinc-protoporphyrin/hour/
milligram of protein. The enzyme kinetic data were analyzed graph-
ically and using the EZ-FIT curve-fitting program on an IBM-PC
microcomputer (Perrella, 1988).
Parameters of the Heme Biosynthesis Pathway-Published proce-
dures were used to 1) record low temperature (liquid nitrogen) ab-
sorption spectra of whole cells, 2) quantify whole cell total heme by
the pyridine hemochrome spectra, 3)analyze the cytochrome content
by reduced-minus-oxidized difference spectra, 4) identify by high
performance liquid chromatography and quantify the porphyrins
accumulated inthe cells and excreted in the culture medium, 5)
measure the intracellular content of 5-aminolevulinic acid (ALA),'
and 6)measure the activity of ALA synthase and coproporphyrinogen
oxidase in cell-free extracts (Urban-Grimal and Labbe-Bois, 1981;
Rytka et al., 1984; Zagorec and Labbe-Bois, 1986). Total non-heme
The abbreviations used are: ALA, 5-aminolevulinic acid; Mes, 4-
morpholineethanesulfonic acid zinc-proto, zinc-protoporphyrin.
bathophenanthroline sulfonate as described by Tangeras et al. (1980),
with minor modifications. Briefly, the samples were "solubilized in
10 mM Mes buffer, pH 4.5, containing 1%(w/v) SDS and incubated
at 95 "C for 1 h in the case of whole cells. Any oxidized Fe(II1) was
reduced with dithionite, and the spectra of the iron complexes were
recorded from 500 to 600 nm, and an absorption coefficient EmM ( A A ,
535-600 nm) = 19.4 was used.
RESULTS
Phenotypic Characteristics of the Mutant Sm12 and Sm41
Strains-The two mutant Sm12 and Sm41 strains were iso-
lated from two different haploid parental strains in a system-
atic search for mutants partially defective in the last steps of
the heme biosynthetic pathway. These mutants accumulate
porphyrins, are photosensitive, fluoresce under UV light, but
maintain sufficient heme synthesis to grow on non-ferment-
able carbon sources such as ethanol or glycerol (Kurlandzka
and Rytka, 1985). Sm12 and Sm41 were shown, by genetic
analysis, to carry single recessive mutations at the HEM15
locus, the structural gene for ferrochelatase. The growth rate
of the mutant cells was lower than that of the parent strain
(Table I). Heme synthesis inthe mutant Sm12 wasabout 2.5-
fold lower than in its parent strain, and itaccumulated large
amounts of porphyrins, mainly protoporphyrin (90-95% pro-
toporphyrin, 2-5% coproporphyrin, and 2-5% pentacarboxy-
porphyrin) (Fig. 1, Table I). The heme content of mutant
Sm41 was also lower than normal, but the relative decrease
could not be quantified since its parent strain is no longer
available. Both mutant cells produced less heme and proto-
porphyrin when growing fermentatively on glucose than when
they were growingrespiratively on ethanol; the reason for the
glucose effect is unclear at present. The majority of protopor-
phyrin accumulated by the mutant cells was, in fact, excreted
at the cell surface and appeared in the culture medium as
small "globules" of (sedimentable) protoporphyrin aggregates.
The addition of Tween 80 or other detergent (Kurlandzka
and Rytka, 1985) to the culture mediumpseudosolubilized
these aggregates, and the excreted protoporphyrin was then
present asthe monomer in the detergent micelles. The spectra
of mutant cells grown in the absence of detergent show two
large absorption bands at 585-590 and 640-645 nm charac-
teristic of aggregated protoporphyrin (Fig. 1).Cells grown in
the presence of 0.2% Tween 80 showed only small peaks,
besides the cytochrome peaks, near 575 and 630 nm for the
protoporphyrin monomer and near 585 nm for zinc-protopor-
phyrin (Fig. 1).Thus, almost all the protoporphyrin made by
the mutant cells in excess of that used for heme synthesis is
excreted out of the cells and very little, if any, is used by
ferrochelatase to make zinc-protoporphyrin.
Table I also shows that the mutant Sm12 contained more
ALA than its parental wild-type strain. This correlates well
with a 2-fold increase in ALA synthase activity. A similar
increase was reported in a mutant partiallydefective in heme
formation due to an altered uroporphyrinogen decarboxylase
activity (Rytka et al., 1984). The regulatory event(s) and the
physiological signal(s) underlying this increase in ALA syn-
thase activity are not clear at present since 1) it is observed
only in partially heme-defective mutants and not in totally
heme-deficient mutants, and 2) the expression of ALA syn-
thase has been shown to be regulated transcriptionally by a
composite array of activation and repression (Keng and Guar-
ente, 1987). Incontrast,the elevated coproporphyrinogen
oxidase activity in Sm12, especially when grown in glucose
was not surprising, since it has been shown that synthesis of
the enzyme is transcriptionally regulated by heme in a nega-
 Yeast Ferrochelatase
Mutations 8543
TABLE I
Characteristics of the heme biosynthetic pathway in the parent (wild-type) and mutant Sm12 and Sm41 strains
The cells were grown in either 2% glucose (Glu) or 2% ethanol + 2% glycerol (Eth) medium and harvested in their exponential phase.
Hemes and ALA were measured on whole cells. Porphyrins were measured both in whole cells and in the culture medium, and the total
(intracellular + excreted) is reported. Porphyrins were 90-95% protoporphyrin in the mutants and8045% coproporphyrin in the parent and
are expressed as such. A L A synthase and coproporphyrinogen oxidase activities were measured in cell-free extracts. All measurements were
performed with the same cell culture. Results given are theaverages for duplicate determinations made in a t least three separate experiments.
The ranges were 20% of the average values. ND: not determined.
Intracellular (/g dry wt) Activity
Strain Carbon source Doubling Total ALA Coproporphyrinogen
time Total hemes porphyrins ALA synthase oxidase
(+excreted)
h nmol pmol nmollhlmg
Wild-type Glu 1.9 80 6 0.25 0.9 2.85
235Eth 3.4 19
0.25 1.6 5.5
Sm12 Glu 2.2 11 2.4
88 1.6 4.6
Eth 5 105 10.5 211 4.5 0.8

Sm41 Glu 1.8 70 32 1.7 ND ND

Eth 3.7 160 60ND 1.2 ND

d A B

n a

WT I FIG.2. Immunodetection offerrochelatase

membrane proteins prepared from the parent and mutant
in total and

strains. Total proteins from trichloroacetic acid treated cells (20 pg)
(panel A ) and membrane proteins (20 pg) (panel B ) were fractionated
by SDS-polyacrylamide gel electrophoresis and transferred to nitro-
Sm 12 cellulose membranes. The membranes were reacted first with yeast
ferrochelatase antiserum and then with alkaline phosphatase-conju-
gated anti-rabbit secondary antibodies. Lane 1 , wild-type strain; lane
2, mutant Sm12; lane 3, mutant Sm41; lane F, isolated ferrochelatase.

HEM15 gene are probably the cause of the enzyme defects.

, . " . I . . . . I " .
5 00 550 600 nm
FIG.1. Low temperature absorption spectra of whole cells
of the parent DCT1-3D (WT) and mutant Sm12 and Sm41
strains. The cells were grown in ethanol + glycerol medium, supple-
mented when noticed with 0.2% Tween 80 (T), and harvested during
exponential growth. Spectra were recorded a t liquid-nitrogen temper-
ature with 40 mg dry weight of cells. Reduction was achieved by
endogenous substrates.
tive fashion (Zagorec and Labbe-Bois, 1986; Zagorec et al.,
1988).
Immunodetection of the Mutant Ferrochelatases-The two
mutants Sm12 and Sm41 made normal amounts of normal
mature-sizedferrochelatase, as estimated by Western blot
analysis of total cell proteins or membrane-bound proteins
(Fig. 2). Normal amounts of full-sized HEM15 mRNA were
also found by Northern blot analysis (data not shown). This
indicates thatthere was noappreciable alterationinthe
processing, structure, or amount of the mutant enzyme pro-
teins and that point mutations in the coding region of the
Identification of the Mutations in the Mutant hem15 Al-
leles-The mutations were identified by sequencing the mu-
tant hem15 alleles in mutant strain DNA after in vitro am-
plification by polymerase chain reaction. The 1.26-kilobase
amplified DNA fragments encompassed the entire HEM15
coding region plus 54 and 26 nucleotides of the 5'- and 3'-
I flanking regions, respectively. A single nucleotide change was
found in each mutant allele. The heml5-3 allele of Sm12
strain containeda C to T transition a t codon 169 that caused
a Ser to Pheamino acid substitution. Thehem15-4 allele of
Sm41 had a T to C transition at codon 174 resulting in a Ser
to Pro amino acid change (Fig. 3). The two mutations are
located near each other andlie in an evolutionarily conserved
region of the proteinrich in hydroxylated amino acid residues.
In particular, the mutation in mutant Sm12 substitutes a Phe
for an invariant Ser (Fig. 3B). These results would suggest
that the two mutations affect ferrochelatase functioning in
the same manner, but toa different extent.
Measurement of the Mutant Ferrochelatase Actiuity-Pre-
liminarymeasurements of the activity of the membrane-
bound mutant ferrochelatases were made by quantifying the
protoheme synthesized under anaerobic conditions from fer-
rous iron (150 PM) and protoporphyrin IX (40 p M ) by its
pyridine hemochromogen spectrum(Camadro and Labbe,
1981). The mutant enzymes were unexpectedly more active
than the wild-type enzyme, 2-3-fold for Sm12 and 1.2-1.5-
fold for Sm41. But assayswithvaryingconcentrations of
a544 Yeast Ferrochelatase Mutations
A
Sm41
Sm12 WT

A C A
G CT A
G CT G T Sm12 Sm41
0.4 ,

I ~ ~ ~ ~ . ~ - ~ . ~ .
Time (min)
FIG. 4. Direct spectrofluorimetric assay for measuring the
Zn2+-protoporphyrinchelatase activity of ferrochelatase in
B membranes of the parent DCT1-3D (WT) and mutant Sm12
. t t .* t * * * . . * and Sm41 strains. The membranes were prepared and the assays
Human 189 T Q - - Q Y - C - - T - S S L N A I Y - Y Y
carried out as under “Materials and Methods.” The reaction mixture
(3 ml final) contained 0.1 M Tris-HCI, pH 7.6,0.02% Tween 80, 1 pM
E.coli 126 P L - - Q F - C - - V - A V W D E L A - I L protoporphyrin IX, 0.5, 0.2, or 0.4mgof protein for the wild-type,
Sm12, or Sm41 strain, respectively. The reaction was initiated by
Yeast 161
+ +
S Q Y P H F S Y S T T G S S I N E L W R Q I
adding membrane protein ( E ) . Zn2+(Zn)was added a t a final con-
centration of 30 p~ for Sm12 and 2 p~ for Sm41. The excitation and
sm12 Sm4 1
emission wavelengths were 418 and 588 nm, respectively.

FIG. 3. Amino acid substitutions in the mutant ferrochela-

tases are located in an evolutionarily conserved region.Panel
A, nucleotide sequences of the polymerase chain reaction-amplified
DNA of the mutants showing the single C to T (codon 169 TCC to
TTC)and T to C (codon 174 TCC to CCC) transitions in the
ferrochelatase hem25 alleles of mutant Sm12 and Sm41 strains,
respectively. Panel B, the sequences of the human and E. coli ferro-
chelatases are from Nakahashi et al. (1990) and Miyamoto et al.
(1991), respectively. Identical amino acids (-) are marked by an
asterisk (*) and conservative ones by a dot (0)above the sequences.
Amino acid substitutions in the yeast mutant enzymes are indicated
by the arrows.

substrates gave inconsistent results. We therefore used a more

sensitive test to analyze the defects of the mutant enzymes
which permitted direct spectrofluorimetric recordingof zinc-
protoporphyrin formation (Camadro and Labbe, 1988). Zn2+ 0.5 1 1.5
can be used in place of Fez+ since 1)the yeast enzyme hasa
similar affinity for both metals,2) there is reciprocal compet- [Protoporphyrin] (PM)
itive inhibition between them, and 3) there is no change in FIG.5. The mutant ferrochelatases have lower affinityfor
the protoporphyrin affinity whatever metal is used (Camadro protoporphyrin and higher V,, than the wild-type enzyme.
and Labbe, 1982, 1988). Thus, both metals appear to bind to Initial velocities (nanomoles of zinc-proto/hour/milligram of protein)
were measured as under“Materials and Methods” and Fig.4, a t
the same site and areused in the same fashionby ferroche- different concentrations of protoporphyrin with a constant concen-
latase. tration of Zn2+:30 p~ for Sm12,lO p~ for Sm41, and endogenous for
The initial velocity of zinc-protoporphyrin formation was the parent ( W T )strain. The curves were obtained with the EZ-FIT
constant for a t least 2-3 min (Fig. 4) and was proportional to program. For clarity, only a few experimental points areshown.
protein concentration in the range used (0.1-1 mg protein/
assay). Tween80 used to pseudosolubilize protoporphyrin was mutant enzymes had lower affinities forZn2+than the normal
kept at 0.2 mg/ml, since higher amounts (0.8 mg/ml) inhibited enzyme (Figs. 4 and 6A). Zn2+had to be added to the reaction
both mutant enzymes: 3-4-fold for Sm12 and 1.2-1.5-fold for mixture for maximal activity of the mutant enzymes, while
Sm41. Both mutant enzymes were totally inhibited by 1 mM the wild-type enzyme was already saturated with the endog-
palmitic acid (20 pl/assay of 20 mg of palmitic acid/ml of enous Zn2+ present in the assay. The two mutant enzymes
dimethyl sulfoxide). This was suerising, and yet unexplained,also had lower affinities for protoporphyrin, and they were
since palmitic acid is required fullforactivity of purified yeast considerably more active,especially Sm12, than the wild-type
and rat ferrochelatases (Camadro and Labbe,1988; Taketani enzyme (Figs. 5 and 6 B ) .
and Tokunaga, 1981) and is often added to ferrochelatase The individual kinetic parameters were obtained from the
assays. Last, we verified thatthenon-hemeferrousiron secondary plots derived from the primary double-reciprocal
presentinthemembranes (14-17 pg/lOO mg of protein), plots of initial rates versus concentration of one substrate at
which can be used in part by ferrochelatase but very slowly various fixed concentrations of the second substrate. They
(Camadro and Labbe, 1982; Tangeras, 1985), was not inhibi- were identical to the values calculated using the “EZ-FIT”
tory to the zinc-chelatase activity; the activity did not change program (Perrella, 1988). The results are reported in Table
in presence of 30 p~ EDTA and excess Zn2+ (100p ~ ) . 11. The high activities of the mutant enzyme were due to
Kinetic Analysis of the Mutant Ferrochelatase-The two increased V,,,,,. The KM for protoporphyrin was increased to
 Yeast Ferrochelatase Mutations 8545
A not directly comparable or related, they correspond surpris-
ingly well with what had been anticipated: similar defects
should affect both mutant ferrochelatases but to different
extents.
The catalytic efficiency (Vm,,/KM) of the Sm12 ferrochela-
tase was decreased in almost the same proportion for the two
substrates.Sinceprotoporphyrinaccumulates in uiuo, it
should not be limiting and it islikely that the concentration
of iron is decisive for heme production. We tried to increase
intracellular ironby growing the cells in the presence of excess
ferric iron (180 PM, which is 18-fold the iron concentrationof
YPE medium). The cells made only half as much protopor-
Ef
0.5 1 1.5
phyrinandtwo-thirds of normal ALA, butthere was no
n ++ (AM)]-l change inheme formation when the whole cells or membrane
preparations were tested for cytochromes and heme content
(data not shown). Heme synthesis in Sm41 cells was also not
increased by iron supplementation. In fact, the concentration

///
of intracellular non-heme iron was only slightly increased in
Sm12 the mutant and wild-type cells growing in iron-enriched me-

t
dium (2.5-3 gmol/g dry weight) as compared to normal me-
dium (2-2.5 wmol/g dry weight), indicating that thecells did
Sm41 not accumulate significantlymore iron when challenged with
V -1 higher extracellular iron concentration. This is because iron
Om6
uptake is limited by the activities of both the ferrireductase
(KM for ferric iron, 3 PM)* and the ferrous iron transporter
(KMfor ferrous iron, 0.15 g~ (Eide et al., 1992)) which are
involved in the reductive assimilation of iron in S. cereuisiae
(Lesuisse and Labbe,1989).

DISCUSSION
10 20 30
We have identified two point mutations in the yeast ferro-
[Protoporphyrin (AM)]" chelatase gene that change two amino acids lying close to-
gether, and we have analyzed their functional consequences
FIG. 6. Double-reciprocal plots of zinc-chelatase activities on enzyme behavior i n uiuo and in uitro. The mutant cells
of wild-type and mutant ferrochelatases as function of sub- synthesized less hemethannormalandaccumulatedand
strate concentration. Initial velocities (nanomoles of zinc-proto/
hour/milligram of protein) were measured at different concentrations excreted large amounts of protoporphyrin. The i n vivo rate of
of ZnZ+with a fixed concentration of protoporphyrin (2 WM) (panel heme synthesis can be estimated from the heme content of
A ) , and at different concentrations of protoporphyrin with a fixed whole cells, the generation time, and knowing that -150 mg
concentration of Zn2+(30, 5, and endogenous for Sm12, ,311141, and of membrane protein isrecovered from 1g dry weight of cells.
wild-type ( WT)enzymes, respectively) (panel B ) . These rates are 0.45, 0.15, and 0.3 nmol heme/h/mg mem-
braneprotein for the wild-type,Sm12 andSm41mutant
TABLE I1 strains, respectively. They represent 4.5, 0.15, and 1%of the
Kinetic parameters of mutant ferrochelatases maximal velocity measured i n uitro forthe zinc-chelatase
The zinc-protoporphyrin activity of wild-type (WT) and mutant activity of the wild-type andmutant enzymes (Table 11).
Sm12 and Sm41 ferrochelatases was measured as in Fig. 6, at different Therefore, the mutant enzymes in Sm12 and Sm41 strains
fixed concentrations of one substrate while varying the concentration function in vivo 30 and 4.5 times less efficientlythan thewild-
of the second substrate. The KM ( p M ) and V,, (nmol Zn-proto/h/mg
of protein) values were calculated graphically from the secondary
type enzyme, which correlates remarkably well with the rela-
double-reciprocal plots and by using the EZ-FITprogram. The results tive increase in their KM for Zn2+ (35 and 4, Table 11). This
are the averages of a t least three determinations with the standard suggests thatironislimiting forheme production in the
deviations. The apparent (app) values for the wild-type enzyme were mutant cells and that no compensatory mechanism increases
measured in this work, while the KM values for ZnZ+were taken from the concentration of iron in the mitochondrial membraneor
Camadro and Labbe (1982,1988) for the yeast membrane-bound and matrix. Iron metabolism and heme synthesis are notdirectly
purified enzyme, respectively. Numbersinbracketsrepresent the
increase with respect to thewild-type values. coupled in Rhodopseudomonas sp. (Moody and Dailey, 1985)
and themouse liver (Tangeras, 1986), but inhibition of heme
KM synthesisstimulatesironuptakemediated by transferrin
Strain V-
Zn2+
Protoporphyrin receptor in rat reticulocytes (Adams et al., 1989).
WT 0.03 app 0.250.15, 9.5 app The amino acid changes, Ser-169to Phe in Sm12 and Ser-
Sm12 1.4 k 0.2 7.5 f 3 100 f 20 174 to P r o in Sm41 mutants, arelocated near each other and
(-45) (-35) (-10) affectferrochelatasefunctioninthesamemannerbutto
Sm41 0.3 f 0.05 0.8 f 0.2 30 f 5 different degree. The functional consequences of the change
(-10) (-4) (-3) of Ser-169, which is conserved among thefive ferrochelatases
known to date, are much more severethan thoseof the change
nearly the same relative extent as was Vmaxfor each mutant. of Ser-174, which is replaced by Val in the bacterialenzymes.
But the KM for Zn2+ was relatively more increased in Sm12 The two mutant enzymes have higherKMfor both substrates,
than in Sm41. Although these changes of the mutant param-
eters areexpressed with respect towild-type values whichare P. Labbe, unpublished results.
8546 Yeast Ferrochelatase Mutations
protoporphyrin and metal. This suggests that the binding
sites of the two substrates are not independentof each other,
in agreement with the model of an ordered sequential enzyme
mechanism where iron binds prior to porphyrin (Dailey and
Fleming, 1983). The mutations may have identified a region
of ferrochelatase that isinvolved in the binding of both
substrates and, probably, they have affected the structure of
the binding sites. This region (Fig. 3B) consists of a central
segment rich in hydroxylated residues which might be part of
the metal-binding domain, flanked on both sides by regions
rich in conservative aliphatic or aromaticresidues that might
be involved in hydrophobic interactions with porphyrin.
The increased V,, of the mutant ferrochelatases, almost
proportional to their increased KM for protoporphyrin, was
surprising. However, a similar situation has been described in
the analysis of porphyrin specificity of ferrochelatase: hydro-
phobic substituents at positions 1, 2, 3, and 4 on pyrroles A
and B lowered both the Vmaxand theKMfor porphyrin of the
enzyme (Honeybourne et al., 1979; Dailey and Fleming, 1983).
This inverse relationship between the affinity for porphyrin
and the rate of heme formation suggests that the release of
heme might control the overall reaction rate. The natures of
the substituents at positions 2 and 4 are important for pro-
ducing a porphyrin either substrate or competitive inhibitor
of ferrochelatase, regardless of its binding ability (Dailey and
Fleming, 1983; Dailey et al., 1989). These substituents also
affect the inhibitory activity of the N-alkyl porphyrins (De
Matteis et al., 1985; Mccluskey et al., 1989). It is significant
that an antibody raised to N-methylmesoporphyrin (2-, 4-
ethyl) did not catalyze the metallation of protoporphyrin (2-,
4-vinyl) or deuteroporphyrin (2-, 4-H) (Cochran and Schultz,
1990). All these results indicate a role for the 2- and 4-vinyl
groups inthe optimal binding of the porphyrin intothe
ferrochelatase active site. We propose that the region of the
enzyme affected by the mutations contributes to the hydro-
phobic interaction(s) and van der Waals contact(s) of the
active site with the porphyrin vinyl group(s).
Detailed analysis of these mutant enzymes for their affinity
for porphyrins substituted at the 2- and 4-positions and for
their inhibition by different isomers of N-alkyl porphyrins,
together with analysis of mutant enzymes obtained by site-
directed mutagenesis of invariant amino acids, will help clarify
the way in which the enzyme binds its substrates, distortsthe
porphyrin ring, and releases heme after chelation of iron.
Acknowledgments-We thank J. Rytka for providing the yeast
mutant strains, J.M. Camadro for assistance with immunological
methods, P. Labbe for helpful advice on the ferrochelatase and iron
assays and 0. Parkes for his help in preparing the manuscript.
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