Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1944-1956 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 12, pp. 2049-2062.

1944

Examination of the Reaction

of Escherichia coli Cytochrome bd-I

in the Fully Reduced State with Cyanide Using

Absorption and Circular Dichroism Spectroscopy

Vitaliy B. Borisov

1,2,a

* and Alexander M. Arutyunyan

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119991 Moscow, Russia

e-mail: bor@belozersky.msu.ru

Received May 27, 2025

Revised June 16, 2025

Accepted June 16, 2025

Abstract— We have earlier begun to investigate the reaction of isolated solubilized dithionite-reduced cyto-

chrome bd-I of Escherichia coli with cyanide [Borisov, V.  B., and Arutyunyan,  A.  M. (2024) The fully reduced

terminal oxidase bd-I isolated from Escherichia coli binds cyanide, J.  Inorg.  Biochem., 259, 112653]. The present

work is a continuation of this study. Using absorption and CD spectroscopy, the following new results were

obtained: (i)  The membrane form of the fully reduced enzyme is also capable of binding cyanide. The apparent

dissociation constant and second-order rate constant values are 81.1 ±  7.8  mM  KCN and 0.11  ±  0.01  M

−1

∙s

−1

respectively. This contradicts the data of other studies according to which the bd-I oxidase located in native

membranes, in the fully reduced state does not bind cyanide. (ii) CO added to the cyano adduct of both

membrane and isolated solubilized forms of the fully reduced cytochrome bd-I displaces cyanide, resulting in

the formation of the CO/enzyme complex. This indicates the reversibility of cyanide binding to the protein.

To saturate the oxidase binding site with CO in the presence of 100 mM KCN, much more CO is required

than upon CO addition to the enzyme not pre-treated with cyanide. CO and cyanide compete for the bind-

ing to the same site in the oxidase (heme d

). Being a stronger ligand, CO “wins” the competition with

cyanide. (iii)  The effect of cyanide on the optical activity of dithionite-reduced cytochrome bd-I was studied.

The CD spectra of the enzyme obtained before and after cyanide treatment indicate that the formation of the

cyano adduct of heme d

leads to a significant weakening of the excitonic interactions between heme  d

and heme b

595

. Schemes of the interaction of cyanide and CO in the presence of excess cyanide with the

enzyme active site are proposed.

DOI: 10.1134/S0006297925601649

Keywords: respiratory chain, terminal oxidase, cytochrome, heme, ligand binding

* To whom correspondence should be addressed.

INTRODUCTION

The processes taking place in aerobic respiratory

electron transport chains of bacteria include a step-

wise oxidation of NADH [E

′  (NAD

/NADH)  ~  –320  mV]

with molecular oxygen [E

′  (O

O)  ~  +820  mV] that

involves several enzyme complexes. Terminal oxidases

are the final portion of respiratory chains. These en-

zyme complexes transfer electrons from ferrocyto-

chrome c or quinol (usually ubiquinol, menaquinol,

or demethylmenaquinol) to O

[1-3]. Enzymatic re-

duction of molecular oxygen to water is accompanied

by the generation of a difference in electrochemical

potentials of hydrogen ions on different sides of the

coupling membrane (ΔμH

) [4-8], which is utilized

by the bacterial cell to synthesize ATP and perform

FERROCYTOCHROME bd-I AND CYANIDE 1945

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

other useful work [9]. Bacterial plasma membrane

contains terminal oxidases from two different super-

families, heme-copper and bd-type (cytochromes bd),

that are different in their primary structure [10-14].

The heme-copper oxidase superfamily includes both

cytochrome c oxidases and quinol oxidases. So far,

all biochemically characterized members of the bd-

type superfamily are quinol oxidases [15, 16]. Unlike

bd-type enzymes, heme-copper oxidases form ΔμH

by acting as proton pumps, which increases their en-

ergy efficiency by 1.5-2 times [17-21]. A lower ener-

gy efficiency of cytochromes bd lacking the proton

pump activity is compensated by their unique physi-

ological properties which are absent in heme-copper

oxidases [22-27]. For example, bd-type oxidases are

more resistant to nitric oxide [28-33], peroxynitrite

[34], hydrogen peroxide [35-40], carbon monoxide

[41-43], sulfide [44-46], ammonia [47], cyanide [44,

48], and some antibiotics [49, 50]. In many pathogen-

ic bacteria, cytochrome bd is a key terminal respi-

ratory oxidase that increases the virulence of these

microorganisms. Genes encoding bd-type oxidases are

absent in the genomes of humans and animals. For

the above reasons, cytochromes bd can be promising

protein targets for the next-generation antibacterial

drugs [51, 52].

The Escherichia coli cytochrome bd-I is often

used as a model bd-type oxidase. It is encoded by

the cydABX operon which is expressed under condi-

tions of O

deficiency [53]. The latter correlates with

a high affinity of cytochrome bd-I for O

[54-56].

According to the structural analysis by single-parti-

cle cryo-electron microscopy (cryo-EM), the bd-I oxi-

dase consists of four subunits: CydA, CydB, CydX, and

CydH/CydY [57,  58] (Fig.  1). CydA contains the Q-loop

that provides quinol binding and oxidation, and three

hemes– one low-spin hexacoordinate heme (b

558

) and

two high-spin pentacoordinate hemes (b

595

and d).

Heme  b

558

serves as a primary electron acceptor in

the oxidation of quinol. Heme  d is a site of oxygen

reduction [59]. It was suggested that in the process

of catalysis, heme  d is converted sequentially from

the oxygenated state to the peroxy, ferryl, and oxi-

dized forms [8, 18, 60, 61]. As a result, within about

1  ms, one oxygen molecule bound to heme  d is re-

duced by four electrons to two water molecules [8,

18]. The role of heme  b

595

in the enzyme functioning

remains obscure. The distance between the iron at-

oms of hemes  b

595

and d (~11  Å [57, 58]) is too large

for these hemes to form a structural binuclear center.

At the same time, the distance between the edges of

these hemes (less than 4  Å [57, 58]) makes van der

Waals contacts between them possible. This is con-

sistent with the experimental data obtained by spec-

troscopy and electrometry [8, 62-74] that indicate the

interaction between hemes  b

595

and d and suggest the

formation of a functional diheme center. Presumably,

the function of heme  b

595

in such a center is rapid

electron transfer to heme d in the process of oxygen

reductase reaction [18,  75,  76].

Cyanide can have a major impact on the bio-

chemistry and physiology of bacteria by inhibiting

aerobic respiration, as well as acting as a signaling

molecule [77]. Cyanide inhibits terminal oxidases

from both superfamilies, but their sensitivity to this

compound varies [1]. The molecular mechanisms of

cyanide inhibition of respiratory enzymes, especially

bd-type oxidases, have not yet been elucidated, which

makes studying the reaction of cytochrome bd with

cyanide especially relevant.

Recently, we found that the isolated solubilized

E.  coli cytochrome bd-I in the fully reduced form

binds cyanide and determined the apparent dissocia-

tion constant (K

  =  52  mM  KCN) and the second-order

rate constant (k

  ~  0.1  M

−1

∙s

−1

) [78]. These data clear-

ly contradict the results of other studies according to

which the bd-I oxidase in the fully reduced state does

not bind cyanide [79, 80]. Moreover, Mitchell et al.

[79] suggested that the inability to bind cyanide in the

fully reduced state is a distinctive feature of bd-type

terminal oxidases. It should be emphasized that the

studies [79,  80] were performed on the cytochrome

bd-I-containing membrane vesicles and not on the iso-

lated solubilized enzyme as in our recent work [78].

Note that the two forms of cytochrome bd-I in these

Fig. 1. Three-dimensional cryo-electron microscopy struc-

ture of E. coli terminal oxidase bd-I at a 3.3 Å resolution

(PDB ID 6RX4). The CydA subunit carries hemes b

558

, b

595

and d. The CydB subunit contains ubiquinone-8 (Q8) and

glycerophospholipid (shown with spheres). Reprinted from

Theßeling et al. [58] under the terms of the Creative Com-

mons Attribution 4.0 International Public License.

BORISOV, ARUTYUNYAN1946

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

studies had different molecular environment, name-

ly bacterial lipids in [79,  80] and detergent micelles

in [78]. There is a growing body of evidence that

the membrane environment can significantly affect

the structure, function, and dynamics of membrane

proteins [81, 82], in particular, terminal oxidases.

As reported in [83], solubilization of bovine heart

submitochondrial particles containing cytochrome  c

oxidase led to a 100 to 1000-fold increase in the af-

finity of the oxidized enzyme for cyanide [83]. Itwas

demonstrated that the membrane environment mod-

ulates the ligand-binding properties of the oxidized

and reduced forms of the E.  coli cytochrome bd-I

toward cyanide and CO, respectively [84, 85]. The

membrane-bound and solubilized forms of the bd-I

oxidase differ significantly in their sensitivity to the

inhibition by CO [41, 42, 86]. In this regard, we de-

cided to study interactions of the membrane form of

the reduced bd-I oxidase with cyanide in order to

elucidate the reason for the discrepancy between the

results of our recent work [78] and data reported in

[79, 80]: whether it could be explained by the differ-

ent environment of the protein or the authors of [79,

80] overlooked the binding of the membrane form of

the reduced cytochrome bd-I with cyanide. The pres-

ent work involved three stages. Having found that

the membrane form of the reduced enzyme was ca-

pable of binding cyanide, first, we repeated on the

membranes the measurements performed before

on the isolated enzyme [78] in order to establish to

what extent the two forms of the bd-I oxidase differ

in the ligand-induced spectral changes and K

and

values. At the second stage, in order to under-

stand whether the observed reaction is reversible

and to obtain confirmation that cyanide binds to the

same site in the protein as CO, we compared spec-

tral changes for the membrane and solubilized forms

of the reduced enzyme titrated with the increasing

concentrations of CO in the presence and absence of

KCN. To our knowledge, no experiments on the ligand

competition for binding to the active site of a bd-type

terminal oxidase have been performed before. At the

third stage, we used CD spectroscopy to investigate

the effect of cyanide on the optical activity of the re-

duced cytochrome bd-I to find out how the binding of

this ligand to heme  d

affects excitonic interactions

between this heme and the high-spin heme b

595

No such studies have been performed before as well.

MATERIALS AND METHODS

Reagents and biological preparations. Sodium

dithionite and carbon monoxide from Merck (Germa-

ny), EDTA, CHES, HEPES from Serva (Germany), and

sodium N-lauroyl sarcosinate from Fluka (Switzer-

land) were used in this work. Other reagents were

of domestic production and of chemically pure grade.

All aqueous solutions were prepared in deionized

water purified with a MilliQ system (Millipore, USA).

CO solution (1  mM) used in the experiments on the

competition of CO and cyanide for the binding to

cytochrome bd-I was obtained by equilibrating de-

gassed water with pure gas at 1  atm at room tem-

perature. To obtain the membrane and solubilized

forms of cytochrome bd-I, the E.  coli strain GO105/

pTK1, which contains a plasmid carrying the corre-

sponding genes for further enzyme overexpression,

was used. This strain also contains a deletion of the

gene coding for the terminal heme-copper quinol ox-

idase bo

that allowed us to obtain cytochrome bd-I

preparations free of the contamination with the bo

oxidase. E. coli cells were grown aerobically in a me-

dium containing 80  mM sodium phosphate, 2.5  mM

sodium citrate, 19  mM ammonium sulfate, 1% tryp-

tone, 0.5% yeast extract, 0.5% casamino acids, 0.01%

L-tryptophan, 2% glycerol, 0.8  mM magnesium sul-

fate, 0.18  mM iron (II) sulfate, 0.1  mM copper (II)

sulfate, 0.005% kanamycin, and 0.01% ampicillin

(pH 7.2). E.  coli membrane preparations (membrane

vesicles) containing cytochrome bd-I were obtained

from bacterial cells using a French press. To isolate

the enzyme, the membranes were solubilized with su-

crose monolaurate. Cytochrome bd-I was purified by

ion-exchange chromatography on a DEAE-Sepharose

Fast Flow column (Sigma, USA). A detailed protocol

for cell growth, isolation of subcellular vesicles, and

solubilization and purification of cytochrome bd-I is

reported in [70]. Cytochrome bd-I concentration was

determined from the difference absorption spectrum

(sodium dithionite-reduced enzyme minus air-oxidized

enzyme) using the millimolar extinction coefficient

Δε

628-607

  =  10.8  mM

−1

∙cm

−1

[65].

Absorption and CD spectroscopy and data

analysis. Optical absorption was recorded with an

Aminco-SLM DW-2000 spectrophotometer (SLM In-

struments, USA) in the spectral (split beam) and

kinetic (dual wavelength) modes. CD spectra were

recorded with a Mark V dichrograph (Jobin Yvon,

France) modified by A.  M.  Arutyunyan. Experiments

were carried out at 21°C under anaerobic conditions

in cuvettes with a 10-mm optical path in the main

incubation medium containing 50mM HEPES, 50 mM

CHES, and 0.5 mM EDTA (pH 8.0). In the case of the

solubilized enzyme, the medium also contained 0.05%

sodium N-lauroyl sarcosinate. To reduce cytochrome

bd-I, an excess of dry sodium dithionite (on a spatula

tip) was added to the cuvette containing the enzyme

and a magnetic stir bar. The resulting reaction mix-

ture was stirred on a magnetic stirrer with the cu-

vette lid closed. The course of cytochrome bd-I reduc-

tion was monitored in real time by recording changes

FERROCYTOCHROME bd-I AND CYANIDE 1947

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

in the absorption spectrum; completion of the re-

duction process was indicated by the cessation of

spectral changes. Concentrated KCN aqueous solution

(4  M) adjusted to pH  8.0 with MES was used to intro-

duce cyanide to the cytochrome bd-I solution.

Data analysis was performed with Origin (Origin-

Lab Corporation, USA) and GIM (Scientific Graphic

Interactive Management System; developed by

A.  L.  Drachev, Belozersky Institute of Physico-Chemi-

cal Biology, Lomonosov Moscow State University).

RESULTS AND DISCUSSION

The addition of KCN to the dithionite-reduced

cytochrome bd-I contained in E.  coli membrane ves-

icles resulted in spectral changes shown in Fig.  2.

The line shapes of the difference absorption spectra

(cyanide  +  dithionite-reduced protein minus dithion-

ite-reduced protein) at different ligand concentra-

tions were similar. The visible region was charac-

terized by the hypsochromic shift of the absorption

band with a maximum at 600 nm and a minimum at

634nm (Fig.  2, curves 1 and 2). In the Soret region, a

W-shaped response with minima at 424 and 443  nm

was detected (Fig.2, curves 1 and 2). Similar spectral

changes were recorded upon the addition of cyanide

to the isolated solubilized enzyme in the same redox

state [78]. This observation, however, contradicts the

data reported in [79, 80] according to which the addi-

tion of cyanide caused no changes in the absorption

spectrum of E.  coli membranes containing the dithi-

onite-reduced bd-I. Since the cyanide-induced spectral

response was relatively small even at the maximum

concentration of the added ligand (ΔA

600-633

  ~  0.015

at 100  mM KCN and 1.55 μM cytochrome bd-I), it is

possible that it was missed by the authors of [79, 80]

due to the low content of cytochrome bd-I in bacte-

rial membranes and/or insufficient sensitivity of the

measuring equipment.

The decrease in the absorption in the region of

the α-band of heme  d

(634  nm) most likely indi-

cated cyanide binding to the heme. Spectral chang-

es around 600  nm, in the region of the α-band of

heme  b

595

, indicated that the ligand binding to the

enzyme led to changes in the heme spectrum. This

might suggest complex formation between heme  b

595

and cyanide. It is known that when cytochromes  a,

b, or c bind a ligand, absorption changes in the

Soret region are at least 5-10 times greater than

those in the visible range of the spectrum [54, 87].

However, in the case of cytochrome bd-I, the cya-

nide-induced responses in the γ-band and the visi-

ble region were comparable in magnitude (Fig. 2,

curves  1 and 2). It could also be assumed that the

resulting cyano adduct had the bridge structure of

the heme  d(Fe

)–C=N–heme  b

595

(Fe

) type. However,

taking into account that the Fe atoms of hemes  b

595

and d are located at a distance of ~11  Å from each

other [57, 58], this is quite unlikely. Therefore, the cy-

anide-induced spectral changes in the membrane form

of the fully reduced bd-I oxidase indicate that the li-

gand binds to heme d and not to the b-type hemes

595

or b

558

). Notably, the cyanide-induced W-shaped

response in the Soret region (Fig.  2, curves 1 and 2)

resembled that observed upon the CO addition [65,

67, 68, 70, 74, 86, 88-90]). In the case of CO, such a

response in the Soret band was explained by the

complex formation between heme  d

and CO accom-

panied by a slight change in the spectrum of heme

595

[68]. Therefore, we assume that the W-shaped

response in the Soret region observed upon cyanide

addition to the membrane-bound cytochrome bd-I

(Fig. 2, curves 1 and 2) also indicates changes in the

absorption spectrum of heme  b

595

caused by the

complex formation between cyanide and heme  d

Fig. 2. Cyanide-induced spectral changes in the membrane

form of fully reduced E. coli cytochrome bd-I. Difference

absorption spectra of bd-I-containing bacterial membranes

obtained by subtracting the spectra recorded after reduc-

tion with dithionite but before addition of cyanide from

the spectra recorded after dithionite reduction and cyanide

treatment. KCN concentration: 10mM (curve1) and 100mM

(curve2). Inset: K

evaluation by analysis of the dependence

of changes in absorbance at 600-633 nm of the membrane

form of fully reduced E.  coli cytochrome bd-I on the cya-

nide concentration; ΔA

600-633

values were determined from

the corresponding difference absorption spectra (KCN  +  di-

thionite-reduced enzyme minus dithionite-reduced enzyme).

To determine K

, experimental data were approximated by a

hyperbola equation using the built-in “Hyperbola function”

in the drop-down menu tab “Analysis → Non-linear Curve

Fit→ Advanced Fitting Tool” of the Origin program. The ap-

parent K

value was 81.1±7.8mM KCN (n =3). The concen-

tration of cytochrome bd-I in the experiments represented in

the main panel and inset was 2.17 and 1.55μM, respectively.

BORISOV, ARUTYUNYAN1948

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 3. Kinetics of the cyanide reaction with the membrane form of fully reduced E. coli cytochrome bd-I. To initiate the

reaction, KCN (final concentration, 30 mM) was added to the cuvette containing the membrane-bound terminal oxidase

bd-I reduced with dithionite. The reaction was recorded in the dual-wavelength mode at 600 nm relative to 633  nm.

The experimental points (curve with noise) were approximated by the single exponential function (smoothed curve) with

obs

  =  0.0064  s

−1

. Inset: k

assessment by analysis of the dependence on the ligand concentration of the pseudo-first-

order rate constant (k

obs

) of the cyanide reaction with the membrane form of fully reduced E. coli cytochrome bd-I. For

this purpose, the experimental points were approximated by the linear function k

obs

 = k

∙[KCN]  +  k

off

[equation for the

pseudo-first-order reaction ([KCN] >> [bd-I]), where k

is the second-order rate constant and k

off

is the dissociation rate

constant] by executing the “Fit Linear” procedure in the drop-down tab of the “Analysis” menu of the Origin program.

The k

value was 0.11  ±  0.01  M

−1

∙s

−1

(n  =  3). Cytochrome bd-I concentration, 2.17 μM.

Figure 2 (inset) shows the dependence of the

absorption changes at 600-633 nm of the membrane

form of the fully reduced E. coli cytochrome bd-I on

the cyanide concentration. Analysis of the obtained

data allowed us to determine the K

value of the

ferroheme d complex with the ligand (81.1  ±  7.8 mM

KCN), which was approximately 1.5 times higher than

the corresponding K

value for the isolated solubilized

enzyme (52 mM) [78]. Presumably, this difference is

due to different lipid and protein environments of the

enzyme. The obtained value was significantly higher

than the K

values for cyanide complexes with the

reduced forms of the E.  coli cytochrome bo

(7  mM)

[79], cytochrome  c oxidase from bovine heart mito-

chondria (0.1-1  mM) [91-94], and horseradish perox-

idase (~1  mM) [95,  96]. In contrast, the affinity of

reduced globins (horse heart myoglobin and human

hemoglobin) for cyanide is much lower, with the K

values of ~0.4  M [97] and 1  M [98], respectively. The

lower affinity of the dithionite-reduced cytochrome

bd-I for cyanide compared to that of the heme-copper

superfamily oxidases may contribute to its relative

insensitivity to this toxic compound [44, 48].

We also studied the kinetics of cyanide reaction

with the membrane form of the dithionite-reduced

bd-I oxidase. Fig.  3 shows a representative kinetic

curve at [KCN]  =  30  mM. Within the studied range

of ligand concentration (10-100  mM  KCN), the reac-

tion rate was well described by the monoexponential

function. The monophasic nature of the reaction in-

dicates the presence of a single ligand binding site

in cytochrome bd-I. This confirms our conclusion

based on the analysis of static absorption spectra

(Fig.  2) according to which only one of the three

hemes forms a complex with cyanide. The binding

rate increased proportionally to the increase in the

ligand concentration (Fig. 3, inset). The second-order

rate constant(k

) was 0.11  ±  0.01  M

−1

∙s

−1

, which was

very close to the k

value obtained for the isolated

solubilized enzyme (~0.1 M

−1

∙s

−1

) [78]). Hence, the

lipid and protein environment most likely does not

affect the binding rate of the reduced bd-I oxidase to

cyanide. Interestingly, the k

values for the cyanide

reaction with the E.  coli cytochrome bo

(572M

−1

∙s

−1

)

[79], cytochrome c oxidase from bovine heart mito-

chondria (35-235  M

−1

∙s

−1

) [91-94], and horseradish

peroxidase (29-115  M

−1

∙s

−1

[95, 96] were much higher.

A relatively low rate of binding of the fully reduced

cytochrome bd-I to cyanide (k

) might explain its

low affinity for the ligand (K

In contrast to cyanide, the addition of CO to

the fully reduced cytochrome bd-I resulted in a ba-

thochromic shift by 5-6  nm of the α-band of heme  d

at 629  nm in the visible region of the absolute ab-

sorption spectrum (Fig. 4c). Accordingly, the CO-in-

duced difference spectrum contained a band with a

maximum at 643-645 nm and a minimum at 622-

625  nm [65, 67, 68, 70, 74, 86, 88-90]. As a result, the

spectral changes induced in the visible region by CO

and cyanide differed significantly (Fig.  4c), allowing

FERROCYTOCHROME bd-I AND CYANIDE 1949

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

us to monitor the competition between these ligands

for the enzyme binding site. The addition of 1  mM

CO to cytochrome bd-I reduced with excess dithion-

ite and then complexed with cyanide by treatment

with 100  mM  KCN resulted in the absolute absorption

spectrum almost identical to that obtained by the ad-

dition of 1  mM  CO to the fully reduced enzyme not

treated with cyanide (Fig.  4c). The observed spectral

changes can be explained as follows: CO added to

the cyano adduct of the fully reduced cytochrome

bd-I displaced cyanide, resulting in the formation of

the enzyme complex with CO with a 80-90% yield.

However, as can be seen from the CO titration curves

(Fig.  4, a and b), much more CO was required to satu-

rate the protein binding site with CO in the presence

of 100  mM  KCN than in the case of untreated cyto-

chrome bd-I. The K

0.5

values in the presence and ab-

sence of 100  mM  KCN were 5.7  ±  0.2 and 0.8  ±  0.1  μM

CO for the membrane form of the oxidase (Fig.  4a)

and 4.4  ±  0.3 and 1.1  ±  0.1  μM CO for the isolated

solubilized enzyme, respectively (Fig.  4b). Therefore,

the data obtained indicate that the binding of cyanide

to the enzyme is reversible. CO and cyanide com-

pete for binding to the same site in cytochrome  bd-I

(heme  d

). Being a stronger ligand, CO “wins” this

competition with cyanide.

The rate constant of the enzyme-cyanide com-

plex decay (k

off

) obtained from the kinetic data

presented in the inset in Fig. 3, was approximately

0.0032  s

−1

(at [KCN]  =  0; according to the equation

obs

 = k

∙[KCN]  +  k

off

). This value was different from

the k

off

value (0.0089  s

−1

) calculated according to the

equation k

off

 = K

∙k

, where K

  =  81.1  mM as deter-

mined from the equilibrium titration data (Fig.  2,

inset) and k

  =  ~0.11  M

−1

∙s

−1

as determined from

the kinetic data (Fig. 3, inset). There are two possi-

ble explanations for this discrepancy. The first one

is that the reaction of cyanide with the protein may

be irreversible, as for example, the reaction of cyto-

chrome bd-I with hydrogen peroxide [99, 100]. How-

ever, this explanation contradicts the results of the

equilibrium titration with CO (Fig. 4) which clearly

indicated the reversibility of the cyanide binding to

the enzyme. Another possible explanation is that the

reaction is reversible but occurs in two stages rather

than one. The binding of cyanide to heme  d

is fol-

lowed by a slower, reversible conversion of the re-

sulting complex into a more stable, spectrally similar

adduct, the decay of which is the rate-limiting step

of the reverse reaction. Further research is needed

to fully elucidate this issue.

Finally, we investigated the effect of cyanide

on the optical activity of the fully reduced cyto-

chrome bd-I. The CD spectrum of the dithionite-re-

duced enzyme (Fig.  5a) was almost identical to that

published by Arutyunyan et al. [69]. The spectrum

showed a small positive signal with a maximum

at 630  nm (Δε

630-653

  ~  21  mM

−1

∙cm

−1

) that coincided

with the absorption maximum of heme  d

in the

same wavelength range. The Soret region contained a

large asymmetric signal consisting of an intense neg-

ative band at 440  nm and a small positive extremum

at 417  nm, with a zero-crossing point at 426  nm.

Fig. 4. The dependence of spectral changes of the membrane

form (a) and isolated solubilized form (b) of fully reduced

E.  coli cytochrome bd-I on the concentration of CO in the

presence and absence of KCN (100 mM). To exclude the

influence of spectral changes caused by the decay of the

cyanide complex with cytochrome bd-I on the CO titration

curves, we used a pair of wavelengths at which such influ-

ence was insignificant: 645 minus 625nm (a) and 643 minus

622 nm (b). To estimate K

0.5

(the concentration of added CO

at which heme d

was half-saturated with CO), the exper-

imental data were approximated by a hyperbola equation

using the built-in “Hyperbola function” in the drop-down

menu tab “Analysis → Non-linear Curve Fit → Advanced

Fitting Tool” of the Origin program. The K

0.5

values in the

presence and absence of 100 mM KCN were 5.7 ±  0.2  μM

CO (n  =  3) and 0.8  ±  0.1  μM CO (n = 3), respectively, for

the membrane form of cytochrome bd-I, and 4.4  ±  0.3  μM

CO (n  =  3) and 1.1  ±  0.1 μM CO (n  =  3), respectively, for the

isolated solubilized enzyme. c) Absolute absorption spectra

of cytochrome bd-I in the fully reduced state (FR) and its

complexes with cyanide (100  mM) and CO (1  mM). The con-

centration of cytochrome bd-I was 2.32  μM (a), 2.53  μM (b),

and 2.67 μM  (c).

BORISOV, ARUTYUNYAN1950

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 5. CD spectra of fully reduced E. coli cytochrome bd-I

and its complex with cyanide. a) Absolute CD spectra of

the enzyme before and after treatment with 200 mM KCN:

curves 1 and 2, respectively. b) Difference CD spectrum ob-

tained by subtracting the spectrum of the enzyme after di-

thionite reduction before KCN addition from the spectrum

after dithionite reduction and KCN treatment. Cytochrome

bd-I concentration, 3.84 μM.

As was previously found [69], the CD spectrum of

cytochrome bd-I is dominated by the optical activi-

ty of ferroheme  d, while individual contributions of

the CD signals of the b-type ferrohemes, b

558

and

595

, are insignificant. The magnitude of the CD sig-

nal in the Soret region (Δε

417-440

  ~  415  mM

−1

∙cm

−1

)

was much higher than that usually observed for the

heme-containing proteins with only one heme or

several hemes weakly interacting with each other

(~10-50  mM

−1

∙cm

−1

) [101, 102]. Such a large magni-

tude of the CD signal of the fully reduced cytochrome

bd-I (Fig. 5a) can be explained by strong electronic

(primarily excitonic and dipole-dipole) interactions

between the electronic transitions of heme  d

and

heme  b

595

in the Soret band [69].

The addition of cyanide to the dithionite-reduced

cytochrome bd-I resulted in the disappearance of the

positive CD signal at 630 nm belonging to heme  d

(Fig. 5a). This was accompanied by a decrease in

the negative CD signal in the Soret region, with the

shifts of the minimum of the negative band from 440

to 442  nm, positive extremum – from 417 to 410  nm,

and zero-crossing point – from 426 to 425  nm

(Δε

410-442

  ~  331  mM

−1

∙cm

−1

(Fig.  5a). Accordingly, the

difference between the CD spectra (dithionite-re-

duced enzyme  +  KCN minus dithionite-reduced en-

zyme, Fig.  5b) revealed a minimum at 630  nm, as

well as an intense positive signal with a maximum

at 437  nm and local minima at 422 and 453  nm

(Δε

437-453

  ~  128  mM

−1

∙cm

−1

). The disappearance of the

positive CD signal at 630  nm caused by the addition

of cyanide indicated that in the fully reduced enzyme,

the ligand binds specifically to the heme  d

iron

and not to the protein moiety near this redox-active

group. The observed pronounced change in the CD

signal magnitude in the Soret region indicates that

the formation of the cyanide–heme  d

complex led to

a significant weakening of the excitonic and, possi-

bly, dipole-dipole interactions between hemes  d

and

595

. This is consistent with the observed changes in

the absorption spectrum of heme  b

595

caused by the

cyanide binding to heme  d

(Fig. 2).

According to the cryo-EM structures of the

E.  coli cytochrome bd-I reported in [57, 58], all the

three hemes and their axial ligands are located in

the CydA subunit. There is a consensus regarding the

nature of the axial ligands of hemes  b: b

558

is coordi-

nated by His186 and Met393, and b

595

is coordinated

by Glu445. However, it is still debated whether the

axial ligand of heme  d is His19  [57] or Glu99  [58].

His19 and Glu99 are located on opposite sides of the

heme d porphyrin ring plane. In the course of oxygen

reductase reaction, O

presumably binds to Fe

heme d on the side where Glu99 is located. According

to the resonance Raman spectroscopy, ferroheme  d in

a complex with cyanide remains in the pentacoordi-

nate high-spin form [103]. If CN

−

binds to the heme

iron on the same side of the macrocycle plane as the

oxygen molecule, two variants of cyanide binding to

heme  d

in the fully reduced cytochrome  bd-I can

be proposed (Fig.  6). If the axial ligand of heme  d

is His19 (variant 1), then the binding of CN

−

to the

heme iron on one side of the porphyrin macrocycle

plane leads to the dissociation of His19 from the oth-

er side of the ring plane. If the axial ligand is Glu99

(variant 2), then it is replaced by CN

−

. In both vari-

ants, heme  d

retains the pentacoordinate high-spin

state after binding with cyanide.

It would also be interesting to discuss a possible

mechanism of CO interaction with the cyano adduct

of heme  d

. It is assumed that in the fully reduced

oxidase, heme  d

in the complex with CO is in the

hexacoordinate low-spin form [65]. Since the absolute

absorption spectra recorded for the CO addition to cy-

tochrome  bd-I pre-treated with 100  mM KCN and un-

treated with cyanide were virtually identical (Fig. 4c),

FERROCYTOCHROME bd-I AND CYANIDE 1951

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 6. Schemes of cyanide binding to unliganded heme d

and CO binding to the cyano adduct of heme d

in fully reduced

E. coli cytochrome bd-I. The central iron atom of heme d

(Fe

), porphyrin ring plane, putative axial ligand of the heme,

and changes in its liganded state are shown (see the text for detailed description of variants 1 and 2). Variant 1 does not

show Glu99 since in none of the putative liganded states of the heme, Glu99 is bound to Fe

it can be assumed that in both cases, the state of

heme  d

in the complex with CO was the same (hex-

acoordinate low-spin). It can also be assumed that,

like O

and cyanide, CO binds to Fe

of heme  d on

the side of the porphyrin macrocycle plane where

Glu99 is located. If so, then regardless of the nature

of heme d protein ligand, the addition of CO leads

to the cyanide dissociation and CO binding to the

heme iron on the same side of the macrocycle plane

and this is accompanied by the binding of His19 on

the other side of the plane (Fig. 6). In this regard,

it should be noted that the difference in the nature

of the heme d axial ligand (His19 or Glu99) revealed

in the two cryo-EM structures of the E.  coli cyto-

chrome bd-I [57, 58] may be explained by the flex-

ibility of the coordination bond between the heme

iron and the ligand, as well as different functional

state of the enzyme in frozen static structures.

Abbreviations

apparent dissociation constant

obs

observed pseudo-first-order binding

rate constant

off

dissociation rate constant

second-order binding rate constant

Acknowledgments

The authors are grateful to R. B. Gennis (University

of Illinois, Urbana, Illinois, USA) for kindly providing

thestrain of E.coli GO105/pTK1.

Contributions

V.B.B. developed the concept and managed the study;

V.B.B. and A.M.A. conducted the experiments, ana-

lyzed and discussed the data, and wrote and edited

the textof the article.

Funding

This work was supported by the Russian Science

Foundation (project no.24-24-00006, https://rscf.ru/en/

project/24-24-00006/).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man or animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

Open access

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tribution 4.0 International License, which permits use,

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in any medium or format, as long as you give appro-

priate credit to the original author(s) and the source,

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statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the

http://creativecommons.org/licenses/by/4.0/.

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