Robert Gennis | University of Illinois at Urbana-Champaign (original) (raw)

Papers by Robert Gennis

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015

The C-family (cbb 3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the ... more The C-family (cbb 3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb 3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb 3 from Vibrio cholerae (CcoNOQP X) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQP X and O 2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c 4 , since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the K C-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

The respiratory heme-copper oxidases catalyze reduction of O 2 to H 2 O, linking this process to ... more The respiratory heme-copper oxidases catalyze reduction of O 2 to H 2 O, linking this process to transmembrane proton pumping. This enzyme family has been classified based on sequence analyses with a focus on residues involved in proton transfer [1,2]. Class A includes oxidases that are found in mitochondria and e.g. in Rhodobacter sphaeroides (aa 3-type), which pump protons with a stoichiometry of 1H + /e −. The B class includes the ba 3 oxidase from Thermus thermophilus, which pumps~0.5H + /e − (reviewed in [3]). This difference in the H + /e − ratios presumably originates from differences in internal e −-H + interactions; while in the A-class oxidases each electron transfer to the catalytic site is linked to proton pumping, in the ba 3 oxidase it is linked to either uptake or release of a "pumped proton". In the A class oxidases the pumping stoichiometry can be modulated in the range 0-1 H + /e − by replacement of single residues along the membrane-spanning proton-conducting (D) pathway. These aminoacid replacements are typically characterized by modulation of the pK a of Glu286 at the end of the pathway. Similar pK a changes were also observed upon replacement of Ser425 (R. sphaeroides, unpublished), located in a protein segment that displays redox-induced structural changes [4,5]. Also with the ba 3 oxidase replacement of single aminoacid residues in or near the (K) proton pathway modulates rates of proton transfer. Interestingly, in this oxidase the choreography of protontransfer events could be altered to specifically block proton uptake to the pump site or to the catalytic site, or release of the pumped proton [3]. Taken together, all these data identified the proton-loading site to be located in a protein segment around Asp372 (ba 3). Furthermore, the data show that a non-integer pumping stoichiometry is an intrinsic feature of the architecture and choreography of the redox-driven proton pump. Abstracts e5 CORE Metadata, citation and similar papers at core.ac.uk

Proceedings of the National Academy of Sciences, 2011

Energy conservation in all kingdoms of life involves electron transfer, through a number of membr... more Energy conservation in all kingdoms of life involves electron transfer, through a number of membrane-bound proteins, associated with proton transfer across the membrane. In aerobic organisms, the last component of this electron-transfer chain is a respiratory heme-copper oxidase that catalyzes reduction of O 2 to H 2 O, linking this process to transmembrane proton pumping. So far, the molecular mechanism of proton pumping is not known for any system that is driven by electron transfer. Here, we show that this problem can be addressed and elucidated in a unique cytochrome c oxidase (cytochrome ba 3 ) from a thermophilic bacterium, Thermus thermophilus . The results show that in this oxidase the electron- and proton-transfer reactions are orchestrated in time such that previously unresolved proton-transfer reactions could be directly observed. On the basis of these data we propose that loading of the proton pump occurs upon electron transfer, but before substrate proton transfer, to t...

FEBS Letters, 2011

The function of membrane‐bound transporters is commonly affected by the milieu of the hydrophobic... more The function of membrane‐bound transporters is commonly affected by the milieu of the hydrophobic, membrane‐spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native‐like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal‐to‐noise value of...

Biochemistry, 2013

The ba 3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Res... more The ba 3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa 3-type oxidases proton uptake to the catalytic site and "pump site" occur simultaneously. However, with the ba 3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than with the aa 3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba 3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though the ba 3 CytcO uses only a single proton pathway for transfer of the substrate and "pumped" protons, the aminoacid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site, respectively. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.

Biochemistry, 2010

Cytochrome ba 3 from T. thermophilus is a member of the B-type haem-copper oxidases, which have l... more Cytochrome ba 3 from T. thermophilus is a member of the B-type haem-copper oxidases, which have low sequence homology to the well-studied mitochondrial-like A-type. Recently, it was suggested that the ba 3 oxidase has only one pathway for proton delivery to the active site, and that this pathway is spatially analogous to the K-pathway in the A-type oxidases. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the timeresolved reaction between fully reduced cytochrome ba 3 and O 2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312→Ser variant in the ba 3 oxidase both reactions associated with proton uptake from solution, the P R →F and F→O transitions, were slowed compared to the wild-type ba 3. The observed time constants were slowed ~3-fold (P R →F, to ~170 μs from 60 μs in wild-type) and ~30-fold (F→O, to ~40 ms from 1.1 ms). In the Thr-315→Val variant, the F→O transition was about 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P R →F transition displayed an essentially unchanged time constant. However, proton uptake from solution was a factor of two slower and decoupled from the optical P R →F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, in strong support for the suggested involvement of the T312 and T315 in proton transfer to the active site during O 2 reduction in the ba 3 oxidase. The ba 3 cytochrome c oxidase (CcO) from Thermus (T.) thermophilus is an integral membrane protein expressed at high temperatures and low oxygen concentrations. The ba 3 CcO is a member of the haem-copper oxidase (HCuO) superfamily, which are terminal oxidases that catalyse reduction of oxygen to water (4 e − +4 H + +O 2 →2 H 2 O) in a sequential mode, i.e. the reaction includes a number of reaction intermediates. The reaction is exergonic and a fraction of its free energy is conserved in the form of a transmembrane † These studies were supported by grants from the Swedish Research Council

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009

The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with t... more The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a 3. It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to ∼ 5-18% of the native level, and the absence of proton pumping in R481Q but retention of ∼ 40% efficiency in R481H and R481L (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl Cf O stretching modes of both the heme a and a 3 in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a 3. In addition to the change in the environments of heme a and a 3 , the Raman data show that the mutations induce a partial conversion of the heme a 3 from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the Cu B-heme a 3 binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.

Isotope Labeling of Biomolecules - Labeling Methods, 2015

Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in ma... more Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.

Biophysical Journal, 2012

The passive transport of small molecules across the plasma membrane is a key physiological proces... more The passive transport of small molecules across the plasma membrane is a key physiological process. Literature measurements of membrane permeability to small molecules have varied widely. We used confocal microscopy to image the transport of molecules into a giant unilamellar lipid vesicle (GUV). Fluorescent dyes were used to trace the transport of molecules. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short exposure. We used this system to study the passive transport of carboxylic acids, which have many properties common to small-molecule drugs. The transport of these acids across cell membranes has been widely studied, but there is much variation in the reported permeabilities. By using pH-sensitive fluorescein-dextran to track the acids permeating through the GUV membrane, our results showed that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive precise permeability values. The permeabilities change with the same trend as oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules. We used the imaging technique described above to study the transport of protons across compositionally asymmetric lipid bilayers. Synthetic asymmetric GUVs were prepared via a microfluidic multiphase droplet flow technology to mimic membrane charge asymmetry. Negatively charged phosphatidylserine was added to an asolectin GUV on either the internal or external leaflet. The permeation rates of protons into and out of these GUVs were measured. The proton distribution across the asymmetric GUV membrane at equilibrium was also studied. This research can reveal how asymmetric cell membrane composition affects small molecule transport behavior in physiological processes.

Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2014

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copp... more The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate ca...

Proceedings of the National Academy of Sciences of the United States of America, Jan 17, 2015

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex t... more The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction ste...

The ubiquinol:cytochrome c 2 oxidoreductase (bc-complex) of Rhodobacter sphaeroides has three mai... more The ubiquinol:cytochrome c 2 oxidoreductase (bc-complex) of Rhodobacter sphaeroides has three main subunits, which bear the prosthetic groups, and contribute to three catalytic sites and internal electron transfer pathways which define the modified Q-cycle mechanism. In this paper, we report on progress in modelling the structure of the bc-complex, and experiments using site directed mutagenesis and biophysical assay to probe the structural and function consequences of specific modifications to these subunits.

Science, 2015

NADPH/NADP(+) (the reduced form of NADP(+)/nicotinamide adenine dinucleotide phosphate) homeostas... more NADPH/NADP(+) (the reduced form of NADP(+)/nicotinamide adenine dinucleotide phosphate) homeostasis is critical for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystallized as a symmetric dimer, with each protomer containing a putative proton channel. The holo-TH is a highly asymmetric dimer with the NADP(H)-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer.

FEBS letters, Jan 2, 2014

Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish... more Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

Gene, 1991

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome ... more The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.

Biochemistry, 2002

and is in contrast to the bovine oxidase, which binds Ca 2+ reversibly. A series of R. sphaeroide... more and is in contrast to the bovine oxidase, which binds Ca 2+ reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca 2+ coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca 2+ ; their spectra are not perturbed by added Ca 2+ or EGTA. The D485A mutant is active, binds to Ca 2+ reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca 2+ binding for both reduced and oxidized states of heme a. The K d value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K d ) 1-3 µM). The rate of Ca 2+ binding with the D485A oxidase (k on ) 5 × 10 3 M -1 s -1 ) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (∼10 -3 s -1 ) and highly temperature-dependent. The k off /k on ratio (190 nM) is about 30-fold higher than the equilibrium K d of 6 nM, indicating that formation of the Ca 2+ -adduct may involve more than one step. Sodium ions reverse the Ca 2+ -induced red shift of heme a and dramatically decrease the rate of Ca 2+ binding to the D485A mutant COX. With the D485A mutant, 1 Ca 2+ competes with 1 Na + for the binding site, whereas 2 Na + compete with 1 Ca 2+ for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na + binding site in bovine COX. No effect of Ca 2+ binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca 2+ plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function. Cytochrome c oxidase (COX) is the terminal enzyme of mitochondrial and bacterial respiratory chains that reduces molecular oxygen to water and conserves the free energy of this exergonic reaction in the form of a transmembrane proton electrochemical potential gradient . The catalytic core of the enzyme is comprised of four redoxactive metal centers (heme a, heme a 3 , Cu A , and Cu B ). In addition, COX contains a number of nonredox metal ions, as revealed first by analytical methods (4-8) and confirmed subsequently by X-ray diffraction studies of the crystal structures (9-13). Zn 2+ is bound to the nuclear-encoded subunit Vb in the bovine COX (9), and Mg 2+ or Mn 2+ is located at the interface between subunits I and II, close to the propionate groups of heme a 3 (9, 14-16). Recent crystallographic studies have drawn attention to the presence and potential significance of a "novel cationbinding site" in subunit I of COX, which can bind to Ca 2+ or Na + . Wikström and his collaborators first discovered that Ca 2+ ions bind reversibly to mitochondrial COX, resulting in a small red shift (1-2 nm) of the absorption spectrum of the reduced heme a and competing with protons for the binding site . These observations were confirmed by other groups (19-21). The binding site was reported initially to be specific for Ca 2+ and protons (18) (or hydronium cations as proposed in 21). Subsequent studies showed that Na + , at physiological concentrations, competes specifically with Ca 2+ and protons for the binding site, although binding of Na + does not induce the spectral shift of heme a (21, 22).

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1992