(Received for publication, November 3, 1995; and in revised form, December 19, 1995)
From the
Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli. Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity. The C207S mutation was also generated in cloned human sulfite oxidase. The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis. The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molybdenum domain. The molybdenum and molybdopterin contents of the two samples were comparable. These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal.
Sulfite oxidase, located in the intermembrane space of animal
mitochondria, catalyzes the oxidation of sulfite to sulfate, the
terminal reaction in the oxidative degradation of the sulfur-containing
amino acids, cysteine and methionine. The enzyme is a dimer of
identical subunits of mass 52 kDa. The N-terminal domain of mass 10 kDa
forms a b-type cytochrome, and the C-terminal
domain of mass 42 kDa anchors the molybdenum cofactor. The molybdenum
cofactor in sulfite oxidase consists of molybdopterin (MPT), (
)a 6-alkyl-dihydropterin containing a unique cis-dithiolene moiety coordinated to molybdenum (1) .
EXAFS studies of rat liver sulfite oxidase have provided evidence for
the presence of 2 Mo=O, 2 to 3 Mo-S, and 1 Mo-O(N)
bonds at the molybdenum center(2, 3) .
The complete
amino acid sequences of sulfite oxidase from chicken(4) ,
rat(5) , and human (6) sources have been reported. In
addition, the amino acid sequences of a related enzyme nitrate
reductase have been reported from a variety of fungal and plant sources (9, 10, 11, 12, 13, 14, 15, 16, 17) . ()(
)Nitrate reductase catalyzes the reduction of
nitrate to nitrite, a critical reaction in the nitrogen assimilation
pathway in fungi and higher plants. The enzyme contains three
prosthetic groups: the molybdenum cofactor, a b
cytochrome, and FAD in binding domains encoded by distinct
segments of the primary sequence. Unlike in sulfite oxidase, the
molybdenum domain of nitrate reductase is at the N terminus, followed
by the central heme domain and the C-terminal flavin domain. The amino
acid sequences of the molybdenum domains of sulfite oxidase and nitrate
reductase are approximately 37% identical, and a single cysteine
residue, corresponding to Cys-207 of sulfite oxidase, is invariant in
all of the sulfite oxidases and nitrate reductases sequenced to date.
It has been postulated that this cysteine functions as a ligand to
molybdenum(4, 6) . Recently it was shown that mutation
of the corresponding cysteine residue in nitrate reductase leads to
loss of activity(18) .
This report describes the site-directed mutagenesis of rat and human sulfite oxidase to generate cysteine to serine mutants for each of the four cysteines in sulfite oxidase. The molybdenum domain of the human sulfite oxidase C207S mutant has been purified, and spectroscopic data indicate that Cys-207 functions as a ligand of molybdenum.
The kinetic mechanism of sulfite oxidase consists of several
steps, including oxidative hydroxylation of sulfite by Mo(VI)
generating Mo(IV), stepwise 1 electron transfers from Mo(IV) to the
heme domain, followed by electron transfer from the heme to the
physiological electron acceptor cytochrome c. Thus, any
mutation causing a loss of activity could be due to an effect on any of
several steps in the reaction pathway and may or may not involve
changes in molybdenum coordination. In order to demonstrate that a
particular residue is a ligand of molybdenum, it is necessary to
demonstrate that site-directed mutagenesis of that residue alters the
absorption spectrum of the MPT-molybdenum chromophore. In the case of
the majority of molybdoenzymes, including sulfite oxidase, it is
virtually impossible to detect changes in the absorption properties of
the molybdenum center owing to the presence of other much stronger
chromophores. In particular, the cytochrome b-type
heme of sulfite oxidase completely masks the absorption spectrum of the
molybdenum center. However, studies using sulfite oxidase purified from
rat liver (19) showed that gentle treatment with trypsin
followed by gel filtration allowed separation of the two chromophores
and isolation of the molybdenum domain. Absorption spectra of the
isolated molybdenum domain revealed certain features of the
MPT-molybdenum chromophore, although total removal of heme was not
achieved in those earlier studies. With the availability of recombinant
sulfite oxidase and techniques for site-directed mutagenesis, it became
possible to examine the effects of specific mutations on enzyme
activity and then to further probe the effects of those mutations that
impair activity by monitoring the absorption features of the molybdenum
center. Changes in absorption properties would be indicative of
disturbances in metal ligation and, along with the effects on enzyme
activity, could be used to identify protein-derived molybdenum ligands.
Tryptic Cleavage of Sulfite Oxidase-Treatment of sulfite oxidase with trypsin has been shown to result in cleavage of the enzyme into two distinct domains, yielding the monomeric heme domain and the molybdenum domain, which remains a dimer(23) . Cleavage of sulfite oxidase results in loss of the sulfite:cytochrome c activity of the holoenzyme; however the molybdenum domain retains the ability to oxidize sulfite to sulfate using ferricyanide as the electron acceptor. Due to extremely low expression of the RSO cysteine mutants, tryptic cleavage of the rat enzyme into its constituent domains was not feasible. When HSO was subsequently cloned it was found to be expressed at a level approximately 5-fold higher than the rat enzyme, suitable for isolation and preparation of the molybdenum domain for spectroscopic characterization. However, HSO proved to be extremely insensitive to trypsin as compared with RSO (Fig. 1A). In order to characterize the molybdenum domain spectroscopically, it is necessary to completely cleave the enzyme, since even small amounts of residual heme domain will interfere with the spectrum of the isolated molybdenum domain. The wild-type human enzyme could be completely cleaved only under conditions which severely degraded the molybdenum domain. Examination of the amino acid sequences showed that HSO contains a lysine rather than an arginine at position 108, the site susceptible to trypsin cleavage. In order to generate an HSO mutant with increased sensitivity to trypsin the lysine at position 108 was altered to arginine by site-directed mutagenesis to create plasmid pRG118-K108R. The sulfite:cytochrome c and sulfite:ferricyanide activities of HSO-K108R were identical to those of wild-type HSO. The HSO-K108R mutant showed greatly enhanced sensitivity to trypsin, allowing cleavage of the holoenzyme under conditions which preserve the activity of the molybdenum domain (Fig. 1B). It is interesting to note that the human K108R mutant was less sensitive to trypsin than the wild-type rat enzyme, suggesting that the R108K substitution is not the sole factor responsible for the reduced trypsin sensitivity of the human enzyme. Plasmid pRG118-K108R-C207S was constructed to express the HSO-C207S mutant in a background that would allow tryptic cleavage of the enzyme and subsequent purification of the molybdenum domain.
Figure 1:
A, sulfite:cytochrome c activity of rat () and human (
) sulfite oxidase during
the course of tryptic cleavage. Trypsin treatment of wild-type
recombinant RSO and HSO was essentially as described by Johnson and
Rajagopalan(23) . Sulfite oxidase, 1 mg/ml in 50 mM Tris-HCl, pH 8.1, was incubated at room temperature for 30 min
with 15 µg trypsin in a volume of 0.5 ml. Aliquots taken for
activity assays were mixed with an equal volume of 50 mM Tris-HCl, pH 8.1, containing 1.5 µg of trypsin
inhibitor/µg of trypsin. B, sulfite:ferricyanide (
)
and sulfite:cytochrome c (
) activity of HSO-K108R
during the course of tryptic cleavage (B). HSO-K108R was
treated with trypsin at room temperature using 100 µg of trypsin/mg
of sulfite oxidase in 50 mM Tris-HCl, pH 8.1. Aliquots taken
for activity assays were mixed with an equal volume of 50 mM Tris-HCl, pH 8.1, containing 1.5 µg of trypsin
inhibitor/µg of trypsin.
Figure 2: HPLC chromatograms of trypsin-treated HSO-K108R (A) and HSO-K108R-C207S (B). Absorbance at 280 nm is represented by the solid line; absorbance at 413 nm is represented by the dotted line. C shows the absorbtion spectrum of the molybdenum domain of HSO (solid line) and HSO-C207S (dashed line). The samples were matched in absorbance at 280 nm.
Figure 3: The proposed structure of the molybdenum center of sulfite oxidase showing the molybdenum atom coordinated to two terminal oxo groups, a third oxygen or nitrogen, the two dithiolene sulfurs of molybdopterin, and the side chain of Cys-207.
The region of the sulfite oxidase and nitrate reductase proteins immediately surrounding Cys-207 is highly conserved, and a consensus sequence TL(Q/V)CAGNRR(S/K)E can be identified containing Cys-207. The molybdenum coordination site consensus sequence was searched for in the sequences of two other human molybdopterin-containing enzymes, xanthine dehydrogenase (24) and aldehyde oxidase(25) . A stretch of similar amino acids was found in xanthine dehydrogenase (TLVSRGTRRTV). This sequence is identical to the consensus sequence in 7 out of 11 amino acids and similar in 9 out 11 positions. In the xanthine dehydrogenase sequence a serine replaces the conserved cysteine. It is possible that serine functions as a ligand to molybdenum in this enzyme and may explain the observed differences in the molybdenum centers of sulfite oxidase and xanthine dehydrogenase. However, no direct evidence for this assumption is available. Aldehyde oxidase, which is 50% identical to xanthine dehydrogenase, shows even less sequence similarity in this specific region. It would seem that the sequences of aldehyde oxidase and xanthine dehydrogenase are too divergent from those of sulfite oxidase and nitrate reductase to allow identification of possible molybdenum coordination sites based on sequence similarities
Recently the x-ray crystallographic structures of two proteins containing molybdopterin cofactors have been reported. The bis(MPT)tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus(7) and the (molybdopterin cytosine dinucleotide) molybdenum-containing aldehyde oxidoreductase from Desulfovibrio gigas(8) . The tungsten atom in the aldehyde ferredoxin oxidoreductase is coordinated to four dithiolene sulfur atoms provided by two MPT molecules. In the D. gigas enzyme the molybdenum atom is coordinated to the dithiolene of a single molybdopterin cytosine dinucleotide. Neither protein contains any protein derived ligands to the metal. The data presented in this paper thus represent the first established instance of the coordination of a protein side chain residue to molybdenum or tungsten in enzymes containing pterin-metal cofactors.
The results of these studies do
not provide evidence for or against the possible coordination of
Ser-207 to the metal in the C207S mutant. Ongoing EXAFS analysis of the
mutant sulfite oxidase should provide the answer to that question.
Comparison of the oxidation-reduction potentials for the Mo(VI)
Mo(IV) and Mo(V)
>Mo(IV) transitions of the mutant molybdenum
center to those of the wild-type enzyme, in conjunction with
stopped-flow spectroscopic studies to determine the effect of the
mutation on the reductive and oxidative half-reactions should provide
an understanding of the basis for the highly attenuated catalytic
activity of the mutant. The successful molecular surgery for separation
of the molybdenum center from the heme domain has made it feasible to
apply spectroscopic techniques such as stopped-flow, magnetic circular
dichroism, and resonance Raman to the detailed analysis of the C207S or
any other mutant sulfite oxidase.