Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Abstract

Cytochrome aa 3 -600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O 2 to 2H 2 O. Cytochrome aa 3 -600 is of interest because it is a very close homologue of the cytochrome bo 3 ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo 3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa 3 -600. Cytochrome aa 3 -600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a menasemiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa 3 -600 in comparison with the ubi-semiquinone in cytochrome bo 3 . Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa 3 -600 in comparison with ubiquinone in cytochrome bo 3 . In addition, the electronic structure of the semiquinone cyt aa 3 -600 is more shifted toward the anionic form from the neutral state in cyt bo 3 .A number of prokaryotes contain heme-copper respiratory oxygen reductases, which utilize a membrane-bound quinol as the substrate (electron donor) (1, 2). These enzymes (quinol oxidases) are closely related to the cytochrome c oxidases, reduce O 2 to water, and also pump protons across the membrane bilayer, generating a proton motive force. The quinol oxidases lack Cu A , which is present in the cytochrome c oxidases, and the amino acid sequences of the quinol oxidases can be distinguished from those of the cytochrome c oxidases by the lack of the Cu A binding motif.The most intensively studied heme-copper quinol oxidase is the cytochrome bo 3 ubiquinol oxidase from Escherchia coli (cyt bo 3 ) 3 (3-8). There are currently over 400 sequences of quinol oxidases that are homologues of cyt bo 3 . The vast majority of these sequences are from proteobacteria (330 sequences) or the firmicutes (80 sequences). Bacillus subtilis, a firmicute, does not contain ubiquinone but relies on menaquinone (see Fig. 1) as a redox component in its aerobic respiratory chain (9). There is a homologue of cyt bo 3 in B. subtilis called cytochrome aa 3 -600, and as expected, this enzyme is a menaquinol oxidase (10 -16). Whereas E. coli cyt bo 3 uses only ubiquinol as a substrate, the B. subtilis cyt aa 3 -600 is strictly a menaquinol oxidase. The motivation of the cu...

Prediction of high- and low-affinity quinol-analogue-binding sites in the aa3 and bo3 terminal oxidases from Bacillus subtilis and Escherichia coli

“…The site with the highest score overlaps with the first sequence motif and should represent the Q H . This hypothesis is also consistent with previous experimental data from EPR studies [39] on B. subtilis QOx.aa 3 . This hypothesis is also consistent with previous experimental data from EPR studies [39] on B. subtilis QOx.aa 3 .…”

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2H and 15N Labeling, HYSCORE Spectroscopy, and DFT Modeling

“…[23] The latter value is the smallest ever reportedf or methyl protons of vitamin Km olecules bound to proteins or dissolved in protic solvents. [16,29,34,35,39,42,63,64] For the symmetric IP 4 -MSK model,t he calculated A iso ( 1 H Me ) % 7.5 MHz is very similar to the experimental value A iso ( 1 H Me ) % 7.8 MHz determined from X-band cw ENDOR spectra of MK 4 C À (4 refers to the number of isoprenoid units of the menasemiquinone radical anion)i n2 -isopropanol. [39] The A iso ( 1 H Me ) value is directly proportional to the unpaired spin density in the SQ p orbital on the adjacent a-carbon.…”

“…Owing to the influence of the nuclear quadrupole interaction, no corresponding peak is detected near n I ( 14 N) in the 14 NH YSCORE spectrum shown in Figure2A. [16,17,19,43] To directly identify the origin of these signals, we constructed histidine and lysine auxotrophsf rom the E. coli JCB4023 strain. Inner membrane vesicles containing overproduced NarGHI werep urified and titrated to generate MSK D in equilibrium conditions.…”

“…Assigning the detected 1 Ha nd 14 Nn ucleit oaparticular chemical group is not straightforward. For this purpose, various strategies have been used, including (i)uniform enrichment of the protein in 2 H [12][13][14] or 15 N [15][16][17][18][19][20][21][22] or of the solvent thankst oH 2 O/ 2 H 2 Oe xchange experiments; [12,[23][24][25][26][27][28] (ii)theoretical calculations of hyperfine and quadrupolar parameters, [13,[29][30][31][32][33] (iii)reconstitution experimentsu sing exogenous quinones with various substituents or chemically labeled with 2 H, [29,34] and (iv) comparison with spectroscopic data obtained on model SQs measured in organic solvents. [35][36][37][38][39] Ad irect and more physiological strategy relies on in vivo isotope labeling of individual amino acids or sets of amino acids,w hich,h owever,r equires engineered auxotrophics trains supplemented with isotopically enriched amino acids.…”

A Caged, Destabilized, Free Radical Intermediate in the Q‐Cycle

“…Most SQ species found in enzymes, e.g. at the Q i site of bc 1 , the Q B sites of bacterial reaction centers and photosystem II, the QH 2 oxidation site of bacterial bo 3 and aa 3 quinol oxidases [32] etc., are bound considerably more tightly than Q or QH 2 , raising the stability constant (by five to ten orders of magnitude), [33] allowing reduction of Q or oxidation of QH 2 to proceed by sequential, one-electron transitions. The high stability constant for these SQ species is critical for the so-called two-electron gating which connects the one-electron chemistry of cytochromes or Fe 2 S 2 with the two-electron chemistry of Q/QH 2 .…”