Research Communication

Novel Flavohemoglobins of Mycobacteria

Sanjay Gupta1, Sudesh Pawaria1, Changyuan Lu2, Syun-Ru Yeh2 and Kanak L. Dikshit11Institute of Microbial Technology, Sector 39 A, CSIR, Chandigarh 160036, India2Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461

Summary

Flavohemoglobins (flavoHbs) constitute a distinct class ofchimeric hemoglobins in which a globin domain is coupledwith a ferredoxin reductase such as FAD- and NADH-bindingmodules. Structural features and active site of heme and reduc-tase domains are highly conserved in various flavoHbs. A newclass of flavoHbs, displaying crucial differences in functionallyconserved regions of heme and reductase domains, have beenidentified in mycobacteria. Mining of microbial genome dataindicated that the occurrence of such flavoHbs might be re-stricted to a small group of microbes unlike conventional fla-voHbs that are widespread among prokaryotes and lowereukaryotes. One of the representative flavoHbs of this class,encoded by Rv0385 gene (MtbFHb) of Mycobacterium tubercu-losis, has been cloned, expressed, and characterized. The ferricand deoxy spectra of MtbFHb displayed a hexacoordinate stateindicating that its distal site may be occupied by an intrinsicamino acid or an external ligand and it may not be involved innitric oxide detoxification. Phylogenetic analysis revealed thatmycobacterial flavoHbs constitute a separate cluster distinctfrom conventional flavoHbs and may have novelfunction(s). � 2011 IUBMB

IUBMB Life, 63(5): 337–345, 2011

Keywords flavohemoglobin; mycobacteria; electron-transfer; phylog-

eny; nitric-oxide dioxygenase.

INTRODUCTION

Flavohemoglobins (flavoHbs) are monomeric proteins having

a classical 3/3 helical globin domain linked with a flavin ade-

nine dinucleotide (FAD)-binding reductase domain that may

transfer electrons to the heme iron and allow its rapid re-reduc-

tion to maintain it in the ferrous state for the rebinding of oxy-

gen (1–3). Rapid electron transfer via redox domain may allow

these two-domain globins to carry out diverse redox reactions

that may be vital for their native host. Recent upsurge in avail-

ability of genomic data from various organisms has indicated

that gene encoding for flavoHb may be widely distributed in

unicellular organisms, both prokaryotes and lower eukaryotes

(1, 2, 4); however, none has been found so far in higher organ-

isms suggesting that flavoHbs may be specifically relevant to

the cellular metabolism of microbes. Physiological function(s)

of flavoHbs has been the subject of intense debate over the last

decade. Several putative functions have been proposed for fla-

voHbs (5–8). However, accumulating experimental evidences

support its role in protection of microorganisms from the delete-

rious effects of nitric oxide (NO) by detoxifying it to nitrate via

the NO-dioxygenation reaction (1, 2, 7, 8). The hydrophobic

distal pocket of flavoHbs has been found much more flexible

and expandable than conventional Hbs (3, 9) that allow them to

accommodate large apolar molecules including phospholipids

and perform other cellular functions. Existence of more than

one flavoHb in some bacteria, yeasts, and fungi (10, 11) suggest

that these hemoglobins (Hbs) may be involved in diverse cellu-

lar functions.

FlavoHbs usually function as an integral part of stress

response and virulence of several pathogenic bacteria and fungi

by maintaining the cell redox homeostasis at the aerobic/anaero-

bic interface when cells are exposed to various environmental

stresses (7, 12). Although importance of flavoHbs for the para-

sitic life has been established in several cases (13, 14), none has

been reported from pathogenic or nonpathogenic mycobacteria

so far. When genomic data of various mycobacterial species was

examined, occurrence of multiple flavoHb encoding genes has

been detected in many of them. After amino-acid sequence

alignment and comparison of mycobacterial flavoHbs, we identi-

fied a novel class of flavoHb, exhibiting unconventional heme

and reductase domains, in mycobacteria apart from conventional

flavoHb. Occurrence of two distinct classes of flavoHbs in

mycobacteria is interesting as well as intriguing. Notably, the

new class of flavoHb is present in majority of mycobacterial

species and it also coexists along with a conventional flavoHb in

Address correspondence to: Kanak L. Dikshit, Institute of Microbial

Technology, CSIR, Sector 39A, Chandigarh 160036, India.

E-mail: kanak@imtech.res.in

Received 17 January 2011; accepted 28 February 2011

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.460

IUBMB Life, 63(5): 337–345, May 2011

some of the mycobacterial species. At present, nothing is known

about mycobacterial flavoHbs and their role in cellular metabo-

lism. This study provides the first report on a new class of

flavoHbs that have been identified in mycobacteria and may not

be widespread among microbes unlike conventional flavoHbs.

Primary characterization of one of the flavoHbs of this group,

present in Mycobacterium tuberculosis, was done, and phyloge-

netic analysis was conducted to understand functional and

evolutionary correlation between two classes of mycobacterial

flavoHbs.

EXPERIMENTAL PROCEDURES

Bioinformatics and Phylogenetic Analysis

BLAST searches within the mycobacterial protein data bank

were done by using the sequences of different flavoHbs, for

example, E. coli, Ralstonia eutropha, and Saccharomyces cere-

visiae. Sequences retrieved from these searches were aligned

using CLUSAL W sequence alignment tool from EMBL-EBI

server. Individual FlavoHb of mycobacteria were identified by

paralogs and ortholog searches using E. coli HMP as primary

template. Multiple sequence alignment and conservation and

divergence within the heme and reductase domains of various

mycobacterial flavoHbs were analyzed. A phylogenetic tree was

built up using the neighbor-joining method (15) focusing on

mycobacterial flavoHbs and their close paralogs/orthologs and

conventional flavoHbs of E. coli, Ralstonia eutropha, Bacillus

subtilis, and Saccharomyces cerevisiae. The evolutionary distan-

ces were computed using the Poisson correction method (16)

and are in the units of the number of amino-acid substitutions

per site. All positions containing gaps and missing data were

eliminated from the dataset (complete deletion option). Phyloge-

netic analyses were conducted in MEGA4 (17).

Bacterial Strains, Plasmids, Gene Cloning,and Protein Purification

E. coli strains, JM 109 and BL21DE3, were used for the

cloning and expression of recombinant proteins. E. coli cells

were grown in Luria Bertani or terrific broth (containing 24 g

of yeast extract, 12 g of Bacto-Tryptone, 12.3 g of K2HPO4, 2.3

g of KH2PO4) at 37 8C at 180 rpm unless mentioned otherwise.

MtbFHb were retrieved from the genomic DNA of M. tubercu-

losis H37Rv and expressed in E. coli using standard polymerase

chain reaction (PCR) techniques. Authenticity of PCR-amplified

gene was checked by nucleotide sequencing. Recombinant

genes were cloned on pET 15b at NdeI –BamHI sites and

expressed in E. coli BL21DE3, cultured in Terrific broth supple-

mented with d-aminolevulinic acid (500 lM) and FeCl3 (20

lM) at 37 8C. Recombinant MtbFHb when expressed in E. coli,

appeared associated with cell membranes and was retrieved af-

ter treating the cell pellet with 1% sarcosyl. Recombinant

MtbFHb was purified from the cell lysate using nickel NTA

column (Quiagen) following manufacturer’s instructions. It

resulted in approximately 70–75% pure preparation of protein

exhibiting distinct reddish pink color. These fractions were fur-

ther purified by ion-exchange column (DEAE-Sepharose CL4B,

Pharmacia), equilibrated with 10-mM TrisHCl (pH 8.0) and

eluted using 0.12 M NaCl gradient. The protein and hemoglobin

profile was monitored at 280 and 414 nm. Absorption and CO

difference spectra of whole cells or purified protein preparation

were recorded using Carry 100 Spectrophotometer as described

previously (18). Gel-filtration chromatography was done on

Superdex 75 column, equilibrated with 50 mM TrisHCl (pH

7.0) and protein was eluted in 0.2 M NaCl after calibration with

standard molecular weight markers (Carbonic anhydrase, 29 kD;

Egg albumin, 40 kD and BSA 66 kD).

NO Consumption and Oxygen Uptake

NO consumption activity of MtbFHb expressing cells was

measured polarographically in a 2-ml reaction chamber with

ISO-NO (World Precision Instruments) essentially as described

previously (19). NO uptake was measured from the slope of

curving traces recorded in the specified concentration of NO

(30 lM) and corrected for background rate of NO decomposi-

tion recorded for the control in the absence of any protein. Ni-

tric oxide dioxygenase (NOD) activity of MtbFHb was deter-

mined following the published procedure (20). Oxygen con-

sumption by cells or cell extract was measured with an oxygen

monitor (Yellow Spring Instrument model 55) at 37 8C in 3 mL

of air saturated 0.1 M potassium phosphate buffer (pH 7.5). The

electrode was calibrated with air equilibrated water. Cell culture

(1 mL) was concentrated, washed with 0.1 M phosphate buffer,

and then added quantitatively to 3-mL air-saturated buffer to

check the oxygen consumption by monitoring the change in the

oxygen concentration of the buffer containing the cells. Three

independent experiments were performed for each set.

RESULTS

Two Distinct Classes of FlavoHbs in Mycobacteria

Computational and sequence analysis of available mycobac-

terial genome and protein data indicated that more than one fla-

voHb may be present in many species of mycobacteria. An

interesting pattern in the occurrence of flavoHb encoding genes

was found in both slow- and fast-growing mycobacteria. Oppor-

tunistic pathogens and fast-growing mycobacteria displayed

presence of two flavoHb encoding genes (Table 1); a conven-

tional flavoHb (type I) similar to other microbial flavoHbs (1,

2) and a new class (type II) showing crucial differences within

the functionally conserved regions of heme and reductase

domains. Interestingly, the new class of type II flavoHb

appeared in majority of mycobacteria including both virulent

and avirulent species. Presence of type II flavoHb in microbial

genome was found restricted to certain bacterial species, mainly

belonging to actinomycetes. Existence of two different flavoHbs

in the same organism suggests that they may be playing differ-

ent functions in cellular metabolism of mycobacteria.

338 GUPTA ET AL.

Structural Characteristics of Type II flavoHbs ofMycobacteria: Unusual Features of Hemeand Reductase Domains

Sequence comparison of mycobacterial type I and type II

flavoHbs with other bacterial flavoHbs (Fig. 1A) indicated

that structural features for adopting a three over three globin

fold and signature sequences of typical microbial globins, for

example, B10-Tyr, CD1-Phe, E7-Gln, F8-His (3, 21), are

present in type II flavoHbs, but there are several differences

within the functionally conserved regions of their heme and

reductase domains. The most notable differences within the

globin domain of type II flavoHb is the lack of conserved

hydrogen bonding and disruption of tetrainteractions between

HisF8-GluH23-TyrG5 and contact between TyrG5 and

TyrH12 within the proximal site due to mutation at GluH23

and TyrH12 residues. These observations indicated that the

peroxidase such as catalytic site, present in conventional fla-

voHbs (22), is absent in this class of flavoHbs. The reductase

domain of type II flavoHbs of mycobacteria is also modified

within the cofactor binding sites, for example, the RXYS

motif of the FAD binding site and GXGXXP motif of the

nicotinamide adenine dinucleotide (NADH) binding sites (23,

24). A RKY/F sequence motif, known as high affinity lipid-

binding motif (25), appeared conserved within the proximal

site of heme in type II flavoHbs. Interestingly, mycobacterial

type II flavoHbs exhibited very high (>70%) overall sequence

conservation among them but displayed less than 25% homol-

ogy with conventional (type I) flavoHbs. These unusual struc-

tural features indicated that type II flavoHbs of mycobacteria

might be structurally and functionally different from conven-

tional flavoHbs.

Cloning, Expression, and Characterization of Type IIFlavoHb from M. tuberculosis

To gain an insight into the primary characteristics of type II

flavoHbs, one of its representatives, encoded by Rv0385 gene

in Mycobacterium tuberculosis, was cloned and expressed in E.

coli. SDS-PAGE analysis confirmed the presence of a 43-kDa

band (Fig. 2A) corresponding to the expected size of M. tubercu-

losis flavoHb (MtbFHb) protein. Gel filtration analysis of

MtbFHb substantiated that it is a monomeric protein of 43.5 kDa

(Fig. 2C). Absolute spectra of MtbFHb indicated that protein pre-

dominantly exists in the ferric state. The absorption spectra of

the ferric species exhibits Soret and visible bands at 414 and

536/570 nm, respectively (Fig. 2B), suggesting a hexacoordinated

low-spin (6CLS) heme with an intrinsic amino acid residue or

exogenous ligand bound to the distal site of the heme. The

absorption spectrum of the ferrous species shows Soret and visi-

ble bands at 428 and 533/559 nm, respectively, substantiating the

6CLS configuration of heme, consistent with the presence of a

sixth ligand. Exposure of the ferrous protein to CO caused the

absorption bands to shift to 423 and 542/572 nm, respectively,

typical for CO-bound heme, indicating that the distal ligand is

displaced by the CO. This is in sharp variance with conventional

flavoHbs that exist in penatcoordinated high spin state (22).

These observations indicated that MtbFHb and presumably other

mycobacterial type II flavoHbs may be structurally and function-

ally distinct from conventional type I flavoHbs.

Table 1

Occurrence and distributions of flavoHbs in mycobacteria

Mycobacterium Species No. of flavoHbs Status

FlavoHbs

Type I (E. coli type) Type II (M. tuberculosis type)

M. laprae 0a Virulent – –

M. tuberculosis (H37 Rv) 1 Virulent – Yes (Rv0385)

M. BovisAF2122/97 1 Virulent – Yes (BCG_0393)

M. abcesseus 1 Virulent – Yes (MAB_4269)

M. paratuberculosis 1 Virulent – Yes (MAP_3851)

M. avium 1 Virulent – Yes (MAV_4795)

M. marinum 1 Virulent – Yes (MMAR_0644)

M. ulcerans 1 Virulent – Yes (MUL_128)

M. gilvum 2 Avirulent Yes (Mflv_4884) Yes (Mflv_0255)

M. smegmatis 2 Avirulent Yes (MSMEG_1336) Yes (MSMEG_0719)

M. vanvallani 2 Avirulent Yes (Mvan_1541) Yes (Mvan_639)

M. sp. JLS 2 Avirulent Yes (Mjls_0944) Yes (Mjls_457)

M. sp. MCS 2 Avirulent Yes (Mmcs0916) Yes (Mmcs_470)

M. sp. KMS 2 Avirulent Yes (Mkms_0933) Yes (Mkms_481)

aPseudogene.

FlavoHbs were identified in different species of mycobacteria using E. coli flavoHb as template followed by its paralogs and orthologs searches. Based on

sequence alignment and comparison, two different classes of flavoHbs were identified. Type I designates for flavoHbs similar to flavoHbs of bacteria and

yeasts. Type II designates for the new class of flavoHbs present in M. tuberculosis and other mycobacteria.

339NOVEL FLAVOHEMOGLOBINS OF MYCOBACTERIA

Figure 1. (A) Structure-based sequence alignment of mycobacterial flavohemoglobins (type I and type II) with E. coli and Ralsto-

nia flavoHbs. Conserved residues in heme and reductase domains of flavoHbs are highlighted in light gray, and the residues, which

are different from conserved and present in MtbFHb (type II), are shown in dark gray. FAD and NADPH binding motifs, present

in conventional flavoHbs, are shown in vertical boxes. E. c, Escherichia coli; R. e, Ralstonia eutropha; M. v, Mycobacterium van-

vallani; M. g, Mycobacterium gilvum; M. j, Mycobacterium ssp. jls; M. s, Mycobacterium smegmatis; M. u, Mycobacterium ulcer-

ans; M. m, Mycobacterium marinum; M. p, Mycobacterium paratuberculosis; M. av, Mycobacterium avium; M. tb, Mycobacterium

tuberculosis; M. a, Mycobacterium absessceu. Type I, denotes protein sequence resembling with conventional E. coli type flavoHbs

and type II, sequence designates for the new class of flavoHbs identified in M. tuberculosis and other mycobacteria. (B) Phyloge-

netic analysis of mycobacterial flavoHbs. The phylogenetic analysis was performed by using MEGA4 (17) program. Mycobacterial

genome was searched extensively for the homologs/paralogs and orthologs for flaoHbs and then the first blast hit of different ge-

nome was selected and analyzed by sequence alignment and comparisons for making the evolutionary tree. Mycobacterial type II

flavoHbs make separate cluster from type I FlavoHbs that form cluster along with conventional flavoHbs of bacteria and yeasts.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

As protection against NO and reactive nitrogen species has

been found one of the major physiological functions of bacterial

and yeast flavoHbs (type I), we tested the NO and oxygen

metabolizing properties of MtbFHb by comparing the NO and

oxygen uptake of MtbFHb overexpressing cells of E. coli.

MtbFHb expressing cells did not show any significant NO

uptake when compared with isogenic cells expressing E. coli

HMP (Table 2) but displayed moderately improved respiratory

activities. NOD activity of MtbFHb was estimated as 12 lM21

s21, which was several folds lower than the HMP of E. coli and

trHbN of M. tuberculosis (26). Overall observations, thus, sug-

gested significant differences in structural and functional proper-

ties of type II flavoHb of M. tuberculosis (MtbFHb) when com-

pared with conventional type I flavoHbs.

Figure 1. Continued.

341NOVEL FLAVOHEMOGLOBINS OF MYCOBACTERIA

Figure 1. Continued.

342 GUPTA ET AL.

Phylogenetic Analysis of Type IIFlavoHbs of Mycobacteria

Coexistence of type I and type II flavoHbs in mycobacteria

led us to speculate that function(s) of these two flavoHbs may

be different from each other. Occurrence of type II flavoHbs in

majority of mycobacteria and their presence in limited number

of bacterial species (mainly actinomycetes, data not shown)

indicated that their function may be novel and specific to their

host. To gain an insight into evolutionary corelation between

type I and type II flavoHbs of mycobacteria, phylogenetic anal-

ysis of two classes of flavoHbs was done. BLAST search within

the microbial genome and protein data bank, using E. coli HMP

and MtbFHb, retrieved FMN reductase of E. coli and cyto-

chrome b5 reductase of Saccharomyces cerevisiae as orthologs

of MtbFHb (type II flavoHbs), whereas benzoate 1,2, dioxyge-

nase appeared one of the closest orthologs of type I flavoHbs of

Figure 2. (A) Overexpression of type II flavoHb of M. tuberculosis in E. coli: (1) Molecular weight marker, (2) E. coli BL21DE3

with control plasmid, pET15b, (3) E. coli BL21DE3 expressing MtbFHb of M. tuberculosis, (4) Purified preparation of MtbFHb.

(B) Spectral properties of MtbFHb. Optical absorption spectra of MtbFHb (ferric, deoxy, and CO bound forms). (C) Gel-filtration

profile of MtbFHb. Molecular mass of MtbFHb was determined on Superdex G-75 gel-filtration column. Molecular Weight refer-

ence standards were taken as 1. Bovine serum albumin, 66 kD, 2. Egg albumin, 40 kD, and 3. Carbonic anhydrase, 29 kD.

Table 2

NO and O2 uptake properties of E. coli expressing MtbFHb

Strains NOa consumption n mole of NO (heme21 s21) O2a uptake (lmol min21 1029 cells)

E. coli RB9060 (D hmp) 0.04 6 0.0 4.8 6 0.64

E. coli BL21DE3 (WT) 1.14 6 0.08 5.3 6 0.49

E. coli BL21DE3 (E. coli HMP) 36.31 6 7.9 5.6 6 0.54

E. coli BL21DE3 (MtbFHb) 5. 32 6 0.07 7. 1 6 0.65

aNO and oxygen uptake measurements of cells, expressing MtbFHb, were done essentially as mentioned in Experimental procedures and also described ear-

lier (19). Before oxygen uptake measurements, the 0.1 M potassium phosphate buffer was stirred vigorously to make it air saturated (DO 5 250 lM). Cell

number in the culture was determined by simultaneously plating the cells on Luria Bertani (LB) plate and counting the colonies. Data represent average val-

ues obtained after three independent experiments.

343NOVEL FLAVOHEMOGLOBINS OF MYCOBACTERIA

mycobacteria. Therefore, a phylogenetic tree was developed by

focusing on type I, type II flavoHbs of mycobacteria and their

first orthologs present in different groups (Fig. 1B). Topology

of evolutionary tree, thus, developed, separated type II flavoHbs

of mycobacteria from type I flavoHbs that formed a separate

group along with conventional flavoHbs of bacteria and yeasts.

Phylogenetically, type II flavoHbs appeared related to electron-

transfer proteins such as FMN-reductase of E. coli and cyto-

chrome b5reductase of Sacchromyces cerevisiae, whereas type I

flavoHbs of mycobacteria and other conventional flavoHbs

exhibited phylogenetic closeness with dioxygenases. Overall

structure of phylogenetic tree and duplication nodes suggest an

ancestral duplication and diversion of type I and type II myco-

bacterial flavoHbs.

DISCUSSION

FlavoHbs are widely spread among bacteria, yeast, and fungi

(1, 2) and represent a unique example of multidomain protein,

where two domains having different functional properties, inter-

act and perform entirely new function(s) by carrying out diverse

redox reactions. Heme and reductase domains of flavoHbs dis-

play significant sequence conservation within their cofactor bind-

ing sites (3, 21). A novel flavoHb, displaying crucial differences

within the functionally conserved regions of heme and reductase

domain, has been identified in many species of mycobacteria.

Occurrence of this unconventional class (type II) of flavoHbs

appeared limited to few microbes, mainly belonging to actinomy-

cetes group. Coexistence of type I and type II flavoHbs in several

mycobacterial species suggest that they may be playing different

functions in cellular metabolism of their host.

Type II flavoHbs may constitute a distinct class within the

flavoHb family and may have novel function(s). Functionally

conserved regions of heme and redox domains of type I fla-

voHbs are modified in type II flavoHbs of mycobacteria sug-

gesting that the interactions of heme and redox domain and

mode of redox reactions are different in these proteins. This is

supported by the observation that type II flavoHb of M. tubercu-

losis exists in hexacoordinate state and lacks NO metabolizing

activity unlike majority of type I flavoHbs. When MtbFHb

(type II) was expressed in E. coli, it appeared strongly associ-

ated with cell membranes and bioinformatics analysis indicated

that its orthologs in E. coli and S. cerevisiae are FMN reductase

and Cytochrome b5 reductse, respectively. Structural and func-

tional properties of type II flavoHbs are not obvious at present;

however, their occurrence in limited group of microbes suggests

that they may have novel function(s) in the cellular metabolism

of their host.

To understand the need for two different classes of flavoHbs

and their evolutionary corelation, phylogenetic analysis of

mycobacterial flavoHbs were conducted. Topology of the evolu-

tionary tree, developed after taking into consideration the paral-

ogs and orthologs of type I and type II flavoHbs of mycobacte-

ria, indicated that type II flavoHbs form a separate cluster from

type I flavoHbs and may be functionally different. However,

both may have originated from a common ancestor and evolved

after duplication and diversion. Although precise origin and

functional roles of mycobacterial flavoHbs remains to be eluci-

dated, respective spacing of mycobacterial type I and type II

flavoHbs and their paralogs suggest that type II flavoHbs may

be phylogenetically more related to electron-transferring pro-

teins (FMN reductases, cytochrome b5 reductases, etc.), whereas

type I flavoHbs are closer to dioxygenases.

The diversity in the number and type of flavoHb-encoding

genes in the genome of mycobacteria might reflect the differ-

ence in requirement of these proteins in individual species.

Type II flavoHbs, thus, may be required for the cellular metabo-

lism of both pathogenic and nonpathogenic mycobacteria as it

has been found in majority of analyzed mycobacterial genome

(except M. leprae, where it has been identified as pseudogene).

Phylogenetic studies on invertebrate globins (27) suggested that

flavoHbs and flavoHb like single-domain Hbs (FHb/SDSgb)

family diverged much more in their structure-function than trun-

cated and globin-coupled sensors. Unconventional flavoHbs of

mycobacteria may add another dimension on diversification of

microbial two-domain globins.

ACKNOWLEDGEMENTS

The authors thank financial support from Council of Scientific

and Industrial Research under the SIP10 grant and NSF grant

095635 (SRY) for carrying out this work.

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