(Received for publication, December 26, 1995; and in revised form, February 12, 1996)
From the
Enzymatic and electron transfer activities have been studied by
polarized absorption spectroscopy in single crystals of both binary and
ternary complexes of methylamine dehydrogenase (MADH) with its redox
partners. Within the crystals, MADH oxidizes methylamine, and the
electrons are passed from the reduced tryptophan tryptophylquinone
(TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c via amicyanin. The equilibrium distribution
of electrons among the cofactors, and the rate of heme reduction after
reaction with substrate, are both dependent on pH. The presence of
copper in the ternary complex is not absolutely required for electron
transfer from TTQ to heme, but its presence greatly enhances the rate
of electron flow to the heme.
Specific protein recognition is of the utmost importance for all
biological systems both for regulation and for transfer of information,
metabolites, and other components of living systems. For electron
transfer, such recognition is needed for proper alignment of donor and
acceptor molecules to achieve efficiency and to prevent energy loss
through chance encounter leading to misdirected electron flow or
abortive complex formation(1, 2) . Several models for
the interaction of protein partners have been developed, largely based
on complementarity of surface charge or surface
topology(3, 4, 5) . However, direct
observation of electron transfer complexes in the crystalline state
between weakly associating partners has been reported in only three
instances. These are a complex between cytochrome c and
cytochrome c peroxidase(6) , a complex between
methylamine dehydrogenase (MADH) ()and
amicyanin(7) , and a ternary complex between the latter complex
and cytochrome c
(8) . Although these
complexes provide much detailed structural information about the
interacting surfaces and arrangement of cofactors between and among the
partners, and suggest potential pathways for electrons to flow during
transfer, questions about their physiological relevance and catalytic
competence do arise. To address these questions, we have undertaken a
single crystal polarized absorption study of the reactivity of the
MADH-amicyanin and MADH-amicyanin-cytochrome c
complexes in their crystalline states.
Polarized absorption
microspectrophotometry can be a useful tool to probe the redox
properties of proteins in the solid state. For example, earlier
microspectrophotometric measurements showed that cytochrome c can diffuse into crystals of yeast flavocytochrome b (L-lactate:cytochrome-c oxidoreductase) to form a reversible and functionally competent
complex(9) . In the present study, spectra have been recorded
of crystals of the MADH binary and ternary complexes prepared using
either copper-containing amicyanin or copper-free apoamicyanin. The
crystals of these holo- and apocomplexes are
isomorphous(10, 11) . This isomorphism is very
advantageous for these microspectrophotometry studies since it provides
an internal control, allowing studies of the reactions of MADH in these
crystalline complexes with and without the possibility of electron
transfer through the copper atom. Furthermore, the spectral components
of the prosthetic groups in this system can be better resolved when
compared in the presence and absence of copper. Thus, this method would
be well suited to test whether the arrangements of electron transfer
partners observed in the two types of crystal lattice are competent for
electron transfer and do not represent merely favorable but accidental
crystal contacts.
MADH catalyzes the oxidation of methylamine in the
periplasm of many methylotrophic and autotrophic bacteria to form
ammonia and formaldehyde concomitant with the two-electron reduction of
its redox cofactor tryptophan tryptophylquinone (TTQ)(12) . In
the autotrophs, the electrons are subsequently passed to a type I
copper protein, amicyanin, then to one or more c-type
cytochromes and finally to a membrane-bound cytochrome oxidase. For Paracoccus denitrificans, cytochrome c has been shown in vitro to accept electrons from a
complex between MADH and amicyanin(13) . The existence of this
complex has been further demonstrated by chemical cross-linking and
steady state kinetic analysis(14, 15) . Rates of the
electron transfer reactions between redox centers in the binary and
ternary complex in solution have been measured by stopped-flow
spectroscopy. The rate for heme reduction by Cu(I) in the ternary
complex is 50-100 s
at 30 °C (16) . The rate for the reduction of copper by MADH in the
binary complex is highly dependent on temperature, reaction conditions,
and whether the substrate-derived amino group remains bound to reduced
TTQ. Measured rates vary from 5 to several hundred per
s(17, 18, 19) .
MADH is an
HL
heterotetramer with subunit molecular masses
of 47 kDa and 15 kDa. The TTQ, located in the small subunit, is derived
from two tryptophan side chains which are cross-linked and further
modified to contain an orthoquinone function through a
post-translational modification(20, 21) . The
amicyanin has a molecular mass of 12.5 kDa and the cytochrome a
molecular mass of 17.5 kDa. In the crystalline binary complex, one
molecule of amicyanin is bound to each half of the MADH heterotetramer
in an identical manner. In the crystalline ternary complex (Fig. 1a), the MADH and amicyanin are related in the
same way as in the binary complex. The cytochrome binds to amicyanin at
the hinge of the
-clamshell on the other side from the MADH
binding site. The MADH-amicyanin interface is formed by a concave
surface on the MADH molecule and a convex surface on the amicyanin. The
latter is made up of one edge of the histidine ligand of the copper
atom surrounded by a patch of 7 hydrophobic surface residues (Fig. 1b). The MADH interface contains the
nonquinolated tryptophan of TTQ surrounded by approximately 8
hydrophobic residues from the small subunit and 4 from the large
subunit. The quinolated tryptophan of TTQ is located in the interior of
the light subunit at the enzyme active site. The distance between the
O-6 quinone of TTQ and the copper is approximately 16 Å while the
closest point on TTQ, C
2, of the second tryptophan is about 9
Å from the copper. The amicyanin-cytochrome c
interface is largely hydrophilic in nature, containing several
ionic interactions, and is smaller than that between MADH and amicyanin (Fig. 1b). The iron of the heme and the copper of
amicyanin are separated by approximately 25 Å and the distance
from the O-6 quinone of TTQ to the iron is approximately 40 Å.
Figure 1:
a, stereo ribbon diagram of the ternary
complex between MADH (half the heterotetramer), amicyanin, and
cytochrome c. The MADH H and L subunits are
shown in gray and green and amicyanin and cytochrome
are shown in blue and yellow, respectively. The three
cofactors, TTQ, copper, and heme, are drawn in black, and the
copper and heme ligands are highlighted in light blue. The
quinone oxygens of TTQ lie close to the center of the
-disk of the
H subunit whereas the second tryptophan ring is exposed to the MADH
surface close to the copper site of amicyanin. The cytochrome approach
is made to the twisted
-strand, which is shared between the two
-sheets, of amicyanin and does not involve MADH. This diagram was
prepared using the molecular graphics program SETOR(32) . b, this stereoview focuses on the two interfaces, one between
amicyanin and MADH and the other between amicyanin and the cytochrome.
The color scheme for the protein chains and cofactors is the same as in a. The copper and heme ligands are in light gray.
Most of the interactions between MADH and amicyanin involve nonpolar
groups, whereas the interactions between amicyanin and the cytochrome
involve main chain hydrogen bonds and a greater number of polar side
chains. Nonpolar groups or polar groups whose aliphatic portions are
within an interface are shown in pink; polar residues which
may be involved in salt bridges or hydrogen bonds are shown as red for acidic and blue for basic. This diagram was prepared
using the molecular graphics program
SETOR(32) .
Figure 2:
Polarized absorption spectra of a single
crystal (0.1 0.05
0.01 mm) of the binary complex
between MADH and apoamicyanin recorded at pH 7.5. In the left panel are the spectra of the crystal oxidized by 3 mM potassium
ferricyanide (since crystals in their native mother liquor were found
to be partially reduced). In the right panel are spectra of
the reduced crystal approximately 5 min after addition of 0.2 mM methylamine. The spectra shown are for the electric vector
polarized parallel (dashed lines) and perpendicular (dotted lines) to the crystallographic c axis. The
isotropic equivalent spectra are also shown (solid lines). The
isosbestic points in the MADH spectrum upon reduction are within
2-3 nm of their values in solution under comparable conditions.
After complete reduction of MADH, a residual peak at approximately 425
nm wavelength, similar to that of the semiquinone form of TTQ, remains.
This peak cannot be eliminated by treatment with dithionite or other
reducing agents and may represent a minor fraction of the enzyme which
has been irreversibly modified.
Addition of methylamine to crystals of the
holobinary complex causes spectral changes indicating the formation of
significant amounts of the semiquinone form of TTQ (Fig. 3). In
these crystals, the semiquinone can only be formed by transfer of one
electron from TTQ to the copper of amicyanin after the TTQ has first
been reduced fully to the hydroquinone form by substrate. This
demonstrates that MADH in the crystalline holobinary complex is
competent both in catalysis and electron transfer. The amount of
semiquinone formed is dependent upon pH. At pH 5.7, a large fraction of
the TTQ remains reduced and a significant absorbance by Cu(II) can be
observed, whereas at pH 9.0 the TTQ is mostly in the semiquinone form.
Furthermore, after the reaction of the crystal with methylamine is
complete, the ratio of semiquinone to reduced TTQ can be shifted
reversibly by shifting the pH, suggesting that the difference between
the redox potentials for the TTQ semiquinone/reduced couple and the
Cu/Cu
couple is pH-dependent.
Figure 3: Isotropic absorption spectrum of a single crystal of the binary complex between MADH and amicyanin approximately 5 min after reduction by 0.2 mM methylamine, observed at pH 5.7 (dashed line), pH 7.5 (solid line), and pH 9.0 (dotted line). The three absorption maxima present in the spectra occur at approximately 330 nm, 420 nm, and 590 nm and correspond to the reduced TTQ, TTQ semiquinone, and Cu(II), respectively. The relative absorbances near 420 nm are not quantitative since they may contain some contribution from a redox inactive species present in the sample (see legend to Fig. 2).
The
pH dependence of the electron distribution between TTQ and the copper
in crystals of the binary complex may result from two factors. One is
stabilization of the TTQ semiquinone at high pH. This has been
demonstrated by solution studies involving titration of MADH with
substoichiometric amounts of methylamine at low ionic strength;
redistribution of electrons between reduced and oxidized MADH to form
semiquinone was found to occur at high pH(31) . This
stabilization could arise, for example, by dissociation of a proton
from reduced TTQ but not from the semiquinone form at high pH. The
other factor could be a pH dependence of the redox potential of
amicyanin when it is complexed with MADH. It is known that the
amicyanin redox potential drops by 73 mV when in complex with
MADH(13) . Reduced amicyanin in the crystalline state has been
found to undergo a conformational change at low pH (below about pH 6)
resulting from protonation of the exposed histidine ligand to copper
with rotation of 180° about the C-C
bond and movement away from the copper into solution. (
)In the binary complex, such a histidine flip would move
the imidazole ring about 0.7 Å closer to MADH, according to a
simple model building experiment, promoting disruption of the
MADH-amicyanin interface. This would destabilize the reduced form of
amicyanin in the complex and diminish its redox potential with respect
to TTQ at lower pH.
Figure 4:
Polarized absorption spectrum of a single
crystal (0.1 0.1
0.03 mm) of the ternary complex
between MADH, amicyanin, and cytochrome c
recorded at pH 7.5. Spectra are recorded for the native complex (solid line) and at 13 min (dashed line) and 60 min (dotted line) after addition of 0.2 mM methylamine.
For this spectrum, the electric vector of the polarized light is
perpendicular to the crystallographic b axis. When the
electric vector is aligned parallel to the b axis, the
spectrum is weak and relatively featureless, consistent with the fact
that the planes of the heme groups which contain the principal
transition dipole moments are approximately perpendicular to the b axis(11) . Since the native crystal was not pretreated
with ferricyanide, it contains a small amount of reduced
heme.
When methylamine is added to the apoternary complex crystal at pH 7.5, very little heme reduction occurs after 1 h, but is nearly complete after 4 days. Conversely, no reduction of the heme occurs at pH 5.7. Since the overall distance from TTQ to heme is the same in the holoternary and apoternary complexes and pathways for electron transfer from TTQ to heme are undoubtedly available within the apoternary complex, the fundamental question arises as to how the pH and the presence of copper in the ternary complex can enhance, by some orders of magnitude, the rate of the electron transfer reaction from TTQ to the heme in the crystalline state.
The reaction rates in these crystalline complexes may be limited by substrate diffusion or by one or more of the catalytic steps and may depend on the local environments of the protein molecules which differ considerably from those in solution. In the case of the holobinary complex, the rate does not appear to be limited by electron transfer from TTQ to copper since no accumulation of reduced TTQ is observed before semiquinone formation. In the case of the ternary complex, the copper to heme electron transfer rate may be rate-limiting. The challenge for the future is to compare rates in the crystal and in solution and to explain the means by which the observed structure might achieve such greater efficiency in solution.
These studies show that the holobinary and the holoternary complexes are competent both for substrate oxidation and for electron transfer. The results do not prove that the orientation of proteins in the crystallized complexes are exactly those which occur in vivo or that this is the only possible orientation for these proteins. The present studies do, however, clearly demonstrate that catalysis and long range electron transfer from TTQ to copper and from TTQ to heme via copper can and do occur in a predictable manner when the proteins are present in this orientation.