Although pre-steady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y.. When WT or F166Y was redu
Trang 1flavocytochrome cellobiose dehydrogenase from the
basidiomycete Phanerochaete chrysosporium
Kiyohiko Igarashi1, Makoto Yoshida1, Hirotoshi Matsumura2, Nobuhumi Nakamura2,
Hiroyuki Ohno2, Masahiro Samejima1and Takeshi Nishino3
1 Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
2 Department of Biotechnology, Tokyo University of Agricultural and Technology, Japan
3 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan
Cellulose is the most abundant natural polymer on
earth, and its degradation is thus an important
compo-nent of the carbon cycle Although cellulose is often
referred to as a b-linked glucose polymer, cellobiose, a
b-1,4-linked glucose dimer, should strictly be regarded
as the repeating unit of cellulose, because adjacent
glucoses show opposing faces to each other in the
cel-lulose chain [1,2] Many microorganisms recognize this
repeating unit and hydrolyze cellulose to cellobiose as
an initial step in the metabolism [3] In filamentous fungi, cellulose degradation had been thought to pro-ceed via two-step hydrolysis, i.e cellulose is hydrolyzed
to cellobiose by various cellulases and the product is further hydrolyzed to glucose by b-glucosidase How-ever, recent cytochemical, kinetic, and transcriptional studies [4–6] have supported another hypothesis
Keywords
cellobiose dehydrogenase; cellulose
degradation; electron-transfer;
Phanerochaete chrysosporium
Correspondence
K Igarashi, Department of Biomaterials
Sciences, Graduate School of Agricultural
and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
Fax: +81 3 5841 5273
Tel: +81 3 5841 5258
E-mail: aquarius@mail.ecc.u-tokyo.ac.jp
(Received 27 February 2005, revised
26 March 2005, accepted 6 April 2005)
doi:10.1111/j.1742-4658.2005.04707.x
Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome con-taining flavin and b-type heme, and plays a key role in cellulose degrada-tion by filamentous fungi To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0 Although pre-steady-state reduction of flavin was not affected by the mutation, the rate
of subsequent electron transfer from flavin to heme was halved in F166Y When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome c reduction occurred syn-chronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome c in both
WT and F166Y These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mech-anism in cytochrome c reduction
Abbreviations
CDH, cellobiose dehydrogenase; F166Y, Phe166Tyr mutant CDH; NHE, normal hydrogen electrode; WT, wild-type CDH.
Trang 2concerning the contribution of cellobiose
dehydroge-nase (CDH; EC 1.1.99.18) to the extracellular cellulose
metabolism of the white-rot fungus Phanerochaete
chrysosporium, and the importance of a combination
of hydrolytic and oxidative reactions in
cellulose-degrading fungi as discussed previously [7–13]
CDH is the only extracellular flavocytochrome
known to be secreted by filamentous fungi during
cel-lulose degradation [11–13] This enzyme carries flavin
and a b-type heme in different domains, and the flavin
domain catalyzes the dehydrogenation of cellobiose
and cello-oligosaccharides to the corresponding
d-lac-tones [8,9,14,15] Although this enzyme was initially
characterized as an oxidase (cellobiose oxidase;
EC 1.1.3.25) [8], its higher affinity for quinones and
ferric compounds than for oxygen [16–18] and the
low-spin character of the heme in both the ferric and
fer-rous states [19] indicate that the electron acceptor of
this enzyme is not molecular oxygen Although many
candidates have been proposed for the electron
accep-tor of CDH, its natural electron accepaccep-tor and the
phy-siological function of the oxidative half reaction of this
enzyme are still uncertain
In a previous kinetic study, we showed the
pre-steady-state two-electron reduction of flavin, followed
by interdomain one-electron transfer from flavin to
heme, resulting in the formation of the flavin
semi-quinone radical and reduced heme [20] In order to
achieve full understanding of the redox reaction of
CDH, the reduction mechanism of the electron
accep-tor should be clarified However, several groups have
proposed two possible mechanisms for this reaction;
electron transfer chain and electron sink mechanisms
[21–23] In the putative electron transfer chain
reac-tion, ferric compounds such as cytochrome c are
reduced by heme after one-electron transfer from
fla-vin, whereas the flavin radical or fully reduced flavin is
an electron donor in the electron sink mechanism In
this study, Phe166, which approaches heme
propion-ate, as shown in Fig 1, was mutated to Tyr, and the
slow interdomain electron transfer mutant F166Y was
produced using the heterologous expression system of Pichia pastoris Presteady-state reduction of cyto-chrome c and re-oxidation of heme in the recombinant wild-type (WT) and mutant enzymes were observed by sequential mixing in a three-syringe stopped-flow spec-trophotometer to clarify the redox mechanism of CDH
Results
Redox properties of WT and F166Y CDH The redox potentials of WT and F166Y were com-pared at various pH values, as shown in Fig 2A The potential of F166Y was lower than that of WT at all
pH values, but the difference was larger at lower pH than at neutral pH The potentials of WT and F166Y were estimated to be 176 and 151 mV vs normal hydrogen electrode (NHE) at pH 4.0 Although the potential of F166Y is 25 mV lower than that of WT, it
is still high enough to receive the electron from reduced flavin, because addition of cellobiose causes spectral changes in F166Y from the oxidized to the reduced form (Fig 2B) Reduced F166Y has typical absorption maxima of CDH at 562, 533 and 428 nm due to a-, b- and c-(Soret) bands, respectively, and the absorption decreased at around 480 nm mainly because of flavin reduction These results are essen-tially the same as those for the WT enzyme [24] and suggest that both flavin and heme are active in the mutant enzyme
Presteady-state and steady-state kinetics
of F166Y The presteady-state reduction of flavin and the subse-quent electron transfer from flavin to heme in WT and F166Y were observed by stopped-flow spectrophoto-metry (Fig 3) The addition of cellobiose caused rapid reduction of flavin, and the absorption at the isosbestic point of heme (449.0 nm) decreased similarly in both
Fig 1 Stereo representation of the heme in CDH and the amino acids within 4 A ˚ of the heme Heme and Phe166 are indicated in color The model was generated from PDB 1D7C [35] using the software PYMOL (DeLano Scientific LLC, San Francisco, CA, USA).
Trang 3WT and F166Y (Fig 3A) The observed rates (kobs) of
WT and F166Y reduction were 53.0 ± 1.0 and
50.0 ± 1.3 s)1, respectively In contrast, an apparent
difference in heme reduction was observed between
WT and F166Y, as shown in Fig 3B In WT CDH,
90% of heme was reduced within 0.1 s, whereas in
F166Y, 13% of heme was still in the oxidized form at
0.2 s after mixing with the substrate The kobs values
for heme reduction of WT and F166Y were
30.2 ± 1.2 and 12.5 ± 0.9 s)1, respectively These
results suggest that only the electron transfer step from
flavin to heme was halved in F166Y, whereas the
ini-tial flavin reduction was not affected by the
muta-tion Presteady-state kinetic experiments for flavin and
heme reduction were carried out at various substrate
concentrations, as shown in Fig 4 The kobsvalues for flavin reduction were identical for WT and F166Y (Fig 4A), whereas those of heme reduction in F166Y was almost half of that of WT at all substrate concen-trations tested (Fig 4B) The dissociation constants of
WT (KWT
d ) and F166Y (KF166Y
d ) obtained from the plots were 107 ± 6.6 and 111 ± 6.5 lm, respectively
As shown in a previous presteady-state kinetic study
of WT CDH, heme reduction was inhibited at high substrate concentrations In this study, this
phenom-A
B
Fig 2 (A) pH dependence of the midpoint potential of heme in WT
(h) and F166Y (d) (B) Absorption spectra of oxidized (solid line)
and reduced (dotted line) forms of F166Y Midpoint potentials of
heme in CDH were measured by cyclic voltammetry as described
in Experimental procedures For the absorption spectra, 2.0 l M
F166Y was scanned with or without 50 l M cellobiose in 50 m M
sodium acetate buffer, pH 4.0.
A
B
Fig 3 Presteady-state reduction of WT and F166Y by cellobiose (A) Time courses of absorption at 449 nm for monitoring flavin reduction (B) Time courses of absorption at 562 nm for monitoring heme reduction Solid line, F166Y; dashed line, WT CDH and cello-biose (final concentrations of 5 and 100 l M , respectively) were mixed in 50 m M sodium acetate buffer (pH 4.0), and the absorption changes were monitored by stopped-flow photometry at 30 C.
Trang 4enon was also observed in F166Y, and the
sub-strate inhibition constant of F166Y (KF166Y
1130 ± 130 lm) was similar to that of WT (KWT
1230 ± 180 lm), reflecting the similar Kdvalues of the
two enzymes The limiting rates of heme reduction for
WT (kWTlim) and F166Y (kF166Ylim ) were 46.9 ± 6.8 and 18.8 ± 1.0 s)1, respectively
The steady-state kinetic parameters are summarized
in Table 1 As expected from the presteady-state experiment, there is no difference in these parameters between WT and F166Y using ubiquinone as an electron acceptor, whereas the mutation affected the kinetic parameters when the redox reaction was mon-itored in terms of cytochrome c reduction The kcat values of cellobiose oxidation monitored in terms of cytochrome c reduction were quite similar to the klim values of heme reduction in both WT and F166Y Interestingly, an increase in cytochrome c concentra-tion inhibited its reducconcentra-tion only in the case of F166Y
Sequential mixing experiment of WT and F166Y
To monitor the redox state of heme in CDH and cyto-chrome c independently in the same reaction mixture, the oxidized and reduced spectra of CDH and cyto-chrome c were compared, as shown in Fig 5 In each hemoprotein, there are four isosbestic points in the 500–600 nm region, where the a- and b-bands of heme absorb We selected 549.0 and 556.7 nm to monitor cytochrome c and heme in CDH, respectively, because these gave the maximum absorption difference
After the initial mixing of WT or F166Y with cello-biose, cytochrome c was added to the reaction mixture and the changes in absorption at 549.0 and 556.7 nm were monitored (Fig 6) The reduction of cyto-chrome c and re-oxidation of heme in CDH were observed synchronously, and the kobs values for cyto-chrome c reduction by WT and F166Y were estima-ted as 662 ± 17 and 643 ± 10 s)1, respectively (Fig 6A,C) i.e almost the same value for the two enzymes The kobs values for the secondary phase, however, were 27.7 ± 2.1 (WT) and 13.3 ± 4.1 (F166Y) These values are quite similar to those of
A
B
Fig 4 Cellobiose concentration dependence of the observed rate
(kobs) for flavin (A) and heme (B) reduction in WT (s) and F166Y
(j) kobsvalues for both prosthetic groups were obtained under the
same conditions as in Fig 3, using 25–500 l M cellobiose as a
sub-strate The fitting of the data was performed as described in
Experimental procedures.
Table 1 Steady-state kinetic constants for WT and F166Y All measurements were carried out at 30 C in 50 m M sodium acetate buffer,
pH 4.0 Cellobiose oxidation was monitored by following the reduction of 1 m M ubiquinone or 50 l M cytochrome c as described in Experi-mental procedures ND, no significant substrate inhibition was observed.
Cellobiose oxidation with electron acceptors
Ubiquinone reduction Cytochrome c reduction
K m
(l M )
k cat
(s)1)
K m
(l M )
k cat
(s)1)
K i
(l M )
K m
(l M )
k cat
(s)1)
K m
(l M )
k cat
(s)1)
K i
(l M )
WT 57.9 ± 7.1 40.1 ± 1.2 28.6 ± 1.6 43.5 ± 0.9 2510 ± 380 326 ± 35 44.2 ± 1.4 1.46 ± 0.12 37.2 ± 0.3 ND F166Y 56.0 ± 5.5 36.8 ± 0.9 17.9 ± 3.0 19.2 ± 0.9 3540 ± 310 293 ± 16 46.4 ± 0.8 0.67 ± 0.11 19.0 ± 0.5 233 ± 42
Trang 5heme reduction in both enzymes at the same cellobiose
concentration As shown in Fig 6B,D, heme in WT
and F166Y remained oxidized during this phase, but
was re-reduced with reduction of cytochrome c The
kobs of cytochrome c reduction depended on the
con-centration of cytochrome c in the region tested (5–
20 lm, data not shown), and was almost identical for
WT and F166Y with limiting values of 1460 ± 140
and 1380 ± 80 s)1, respectively
Discussion
Two mechanisms, electron transfer chain and electron sink, have been proposed for the redox reaction of CDH, and several previous kinetic studies have attemp-ted to clarify the overall reaction of this enzyme [21–23] However, uncertainty remains, possibly because of the special features of this enzyme The optimum pH values
of flavin reduction by cellobiose (pH 4.5–5.0) and of electron transfer from flavin to heme (pH 3.5–4.0) differ from each other, and the rate-limiting step of the reac-tion thus depends on the pH of the reacreac-tion mixture [20,25] Moreover, a higher concentration of substrate (cellobiose) inhibits presteady-state heme reduction, but not flavin reduction [20,26], suggesting that binding of substrate to the active site of the flavin domain inhibits electron transfer from flavin to heme This phenomenon makes it difficult to solve the redox mechanism of this enzyme using kinetic results obtained with only WT CDH In this study, therefore, we compared the ther-modynamic and kinetic features of recombinant WT and the slow electron transfer mutant F166Y
The presteady-state electron transfer from flavin to heme was halved in F166Y compared with WT This was expected from the thermodynamic result that the redox potential of heme in F166Y was lower than that
of WT However, when the electron transfer rate k (s)1) and the driving force DG (eV) were analyzed in terms of electron transfer theory, which was recently developed by Dutton’s group [27], the edge-to-edge distance R and the reorganization energy k of F166Y were higher than those of WT when one of these parameters was fixed and used for the calculation (data not shown) This indicates that the halved electron transfer rate of F166Y is due not only to thermody-namic factors, but also involves kinetic changes in this mutant; for example, the change of Phe to Tyr may change the charge of the protein surface, resulting in a change in the interaction between the two domains
This would produce a higher R or k value for F166Y
compared with the WT enzyme That only F166Y shows significant substrate (cytochrome c) inhibition might be because of the weaker domain interaction of this mutant enzyme The F166Y mutant is, however, useful for monitoring the electron transfer step of CDH, because the mutation affects only the electron transfer step from flavin to heme, but not initial flavin reduction or cytochrome c reduction
WT or F166Y was first mixed with cellobiose, and then the cellobiose–CDH mixture was mixed with cyto-chrome c after 0.1 s (WT) or 0.2 s (F166Y) At the second mixing time, 90% of heme is in the ferrous state and the flavin forms a semiquinone, as confirmed
A
B
Fig 5 Absorption spectra of heme in CDH (A) and cytochrome c
(B) for comparison of the isosbestic points The spectra of the
oxi-dized (solid line) and reduced (dashed line) forms are compared in
the range of 450–650 nm Dotted lines at 549.0 (left) and 556.7
(right) show the isosbestic points of heme in CDH and
cyto-chrome c, respectively.
Trang 6previously by EPR [20], suggesting that most of the
enzyme is in the two-electron reduced form In previous
studies, prereduced CDH was often used to observe the
electron transfer from heme to cytochrome c with
cello-biose or ascorbate as an electron donor [22,23,28]
With-out a sequential mixing technique, however, it is difficult
to monitor cytochrome c reduction, because premixing
with the electron donors produces one- (ascorbate) or
three- (cellobiose) electron-reduced CDH, but not the
two-electron-reduced form with flavin radical and
reduced heme Soon after the two-electron-reduced
CDH was mixed with cytochrome c, synchronous
cyto-chrome c reduction and re-oxidation of heme were
clearly observed in WT and F166Y with similar
bio-molecular rate constants (6· 106m)1Æs)1) This indicates that, at the initial phase, the electron is transferred from heme to cytochrome c In contrast, the second phase of cytochrome c reduction was not concentration depend-ent, but depended on electron transfer from flavin to heme Considering that the kcatvalue for cytochrome c reduction was almost identical to the klimvalue for heme reduction in both WT and F166Y, the two electrons in reduced CDH are sequentially transferred from reduced flavin to cytochrome c via heme Cameron and Aust [22] recently demonstrated that the reduction rate of cyto-chrome c by fully (three-electron) reduced CDH was lower than the rate under steady-state turnover, and suggested that the flavin radical reduces cytochrome c
Fig 6 Time courses of redox state of heme in WT (A, B) and F166Y (C, D) after sequential mixing with cellobiose and cytochrome c Absorptions at 556.7 (solid line) and 549.0 (dashed line) nm were used for monitoring the heme in CDH and cytochrome c, respectively WT
or F166Y (5 l M ) was mixed with 100 l M cellobiose, and 20 l M cytochrome c was then added and mixed after 0.1 s (WT) or 0.2 s (F166Y) using a sequential mixing stopped-flow apparatus The absorption changes were monitored after mixing with cytochrome c, and the initial (0–0.020 s) and secondary (0–0.2 s) phase are seen in left (A, C) and right (B, D) panels, respectively Conditions: 50 m M sodium acetate buf-fer (pH 4.0) at 30 C.
Trang 7In the same report, moreover, they proposed that the
heme in CDH acts as an electron sink, because Rogers
and co-workers reported that heme is oxidized during
steady-state cytochrome c reduction [28] Although this
phenomenon was also observed in the second phase of
presteady-state cytochrome c reduction, it is because of
the significant difference between the electron transfer
rate from flavin to heme (30 s)1) and that from heme to
cytochrome c ( 1500 s)1) After the electron is loaded
from flavin to heme, it is transferred to cytochrome c
without any significant time lag Consequently, all the
results obtained in this study apparently indicate that
the overall redox reaction of CDH occurs through
the electron transfer chain mechanism, as shown in
Scheme 1
Although this study clearly demonstrates an electron
transfer chain mechanism of CDH when cytochrome c
is used as an electron acceptor, it is too early to
con-clude that the mechanism is also used during cellulose
degradation by the fungus because its natural electron
acceptor is still uncertain Considering the kinetic
effi-ciency of cytochrome c for CDH is quite high
com-pared with other ferric compounds, it is possible that
the filamentous fungi produce a cytochrome c-like
hemoprotein extracellularly and utilize it as an electron
acceptor of CDH Indeed, there are several
hypo-thetical proteins with a secretion signal and a
cyto-chrome c-binding motif encoded in the total genome
sequence of P chrysosporium [29] To clarify the true role of CDH, therefore, it is important to consider the interaction with other extracellular redox proteins As demonstrated in our kinetic studies, including this work, the redox reaction of CDH is regulated by cellobiose concentration and pH at the interdomain electron transfer step This might provide a clue to identify the redox system of CDH in vivo
Experimental procedures
Materials
d-Cellobiose was purchased from ICN Biomedicals (Irvine,
CA, USA) Ubiquinone (2,3-dimethoxy-5-methyl-1,4-benzo-quinone) and bovine heart cytochrome c were purchased from Wako Pure Chemical Industries (Osaka, Japan) To assess the pH dependence of the mid-point potential of heme,
50 mm buffers were used as described previously [20,30]
Steady-state enzyme assays
To obtain the steady-state kinetic parameters of cellobiose oxidation, the reduction rate of 1 mm ubiquinone or 50 lm cytochrome c was plotted against concentration of cello-biose (0–1 mm) Reduction rates for ubiquinone and cytochrome c were also measured at various concentrations (0–1 mm for ubiquinone and 0–50 lm for cytochrome c) using 500 lm cellobiose as a substrate The reductions of ubiquinone and cytochrome c were monitored photometri-cally at 406 nm (De406¼ 0.745 mm)1cm)1) and 550 nm (De550¼ 17.5 mm)1cm)1), respectively Because apparent substrate inhibition was observed when cytochrome c was used as an electron acceptor, the obtained substrate dependence plots were fitted to the Michaelis–Menten equa-tion with a substrate inhibiequa-tion constant (Ki) Unless other-wise noted, steady-state kinetic parameters (Km and kcat) were estimated by nonlinear fitting of the data to the Michaelis–Menten equation using deltagraph v 5.5 (SPSS Inc., Cary, NC, USA and Redrock Software, Inc., Salt Lake City, UT, USA) and kaleidagraph v 3.0.8 (Synergy Software, Reading, PA, USA)
Preparation of wild-type CDH and F166Y mutant Recombinant wild-type P chrysosporium CDH was hetero-logously expressed in the methylotropic yeast Pichia past-oris and purified as described previously [24] Site-directed mutagenesis was carried out based on the overlap extension and nested primers methods, as described elsewhere [31–34] Synthetic oligonucleotides, F166Y-F, 5¢-CACAC
underlined); AP1-EcoRI-F, 5¢-TTTTCAGCGTTCTCGGA ATTCCAGAGTGCCTCACAGTTTACCGAC-3¢; AP1-F,
Scheme 1 Proposed catalytic cycle of CDH using cellobiose and
cytochrome c as electron donor and electron acceptor, respectively.
CB, cellobiose; CBL, cellobionolactone; F, flavin; H, heme; ox,
oxi-dized form; red; reduced form; sq: semiquinone form.
Trang 85¢-TCAGCGTTCTCGGAATTC-3¢; AP2-Xba-R, 5¢-TTTT
ACAGTAATATAAAGAATTTCGCTCTAGATCAAGGA
CCTCCCGCAAGCGCGAG-3¢; and AP2-R, 5¢-TTACA
GTAATATAAAGAATTTCGCTCTAGA-3¢, were used to
obtain the nucleotide fragment of F166Y (f166y) The
frag-ment was subcloned into the pCR4Blunt-TOPO vector
(Invitrogen, Carlsbad, CA, USA) as described in the
manu-facturer’s instructions, and the vector pCR4
Blunt-TOPO⁄ f166y was digested with EcoRI and XbaI (TaKaRa
Bio, Shiga, Japan) and ligated into the pPICZa-A vector
(Invitrogen) at the same restriction sites The vector
pPICZa-A⁄ f166y was then linearized with Bpu1102I
(TaKaRa Bio) and transformed into Pichia pastoris
KM-71H using a MicroPulser electroporation device (Bio-Rad
Laboratories, Hercules, CA, USA) The Zeocin-resistant
transformant was cultivated in a growth medium (1% yeast
extract, 2% polypeptone, 1% glycerol; w⁄ v) for 24 h at
30C, followed by the induction medium (1% yeast
extract, 2% polypeptone, 1% methanol; w⁄ v) for 48 h at
26.5C, and F166Y was purified from the culture filtrate
with the same protocol as described previously [24] The
purity of wild-type CDH and F166Y was confirmed by
SDS⁄ PAGE and by the absorption spectrum
Measurement of midpoint potential of heme
in CDH
A direct electrochemical technique was used to measure the
mid-point potential according to our previous report [30]
Glassy carbon, platinum, and Ag⁄ AgCl were used as the
working, counter, and reference (+205 mV vs NHE at
25C) electrodes, respectively Cyclic voltammetry was
performed in the presence of 50 mm MgCl2 using an ALS
Electrochemical Analyzer 624A, and the potential was
deter-mined by averaging the anodic and cathodic peak potentials
Presteady-state kinetic studies
Presteady-state reduction of flavin and heme was monitored
at the appropriate isosbestic point using an Applied
Photo-physics SX-18MV kinetic spectrophotometer and the
observed rate (kobs) was estimated by fitting to the double
exponential curve (flavin reduction) or single exponential
curve with lag (heme reduction) according to our previous
report [20] Because apparent substrate inhibition was
observed in heme reduction, the substrate inhibition
con-stant (Ki) was used to estimate the limiting rates (klim) of
heme reduction as described previously [20] Rapid
reduc-tion of cytochrome c by reduced CDH and re-oxidareduc-tion of
heme in CDH were monitored with the same equipment,
but using a sequential mixing mode as follows Wild-type
CDH and F166Y were first mixed with cellobiose, and the
solution was then mixed with cytochrome c at 0.1 s (WT)
or 0.2 s (F166Y) after the initial mixing, when almost 90%
of heme was reduced Final concentrations of WT, F166Y,
cellobiose and cytochrome c were 5.0, 5.0, 100 and 5–
20 lm, respectively To monitor the reduction of heme in CDH and cytochrome c independently, absorption at the isosbestic points, 549.0 nm for cytochrome c (De549.0¼ 16.8 mm)1Æcm)1) and 556.7 nm for heme in CDH (De556.7¼ 9.29 mm)1Æcm)1), was monitored All presteady-state measurements were carried out at least three times in
50 mm sodium acetate buffer pH 4.0 at 30C, and the data were analyzed as described previously [20]
Acknowledgements
This research was supported by Grants-in-Aid for Sci-entific Research to KI (No 15780206), MS (No 14360094), and TN (No 16205021), and by Grant-in-Aid for Scientific Research on Priority Area to TN (No 12147208) from the Ministry of Education, Cul-ture, Sports, Science and Technology, and a Research Fellowship to MY (No 08446) from the Japan Society for the Promotion of Science
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