Relation between interface properties and kinetics of electron transfer in the interaction of cytochrome f and plastocyanin from plants and the cyanobacterium Phormidium laminosum

Cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum react an order of magnitude faster than their counterparts from chloroplasts when long-range electrostatic interactions have been screened out by high salt concentration [Schlarb-Ridley, B. G., et al. (2002) Biochemistry 41, 3279-3285]. To investigate the relative contributions of the reaction partners to these differences, the reactions of turnip cytochrome f with P. laminosum plastocyanin and P. laminosum cytochrome f with pea plastocyanin were examined. Exchanging one of the plant reaction partners with the corresponding cyanobacterial protein nearly abolished electron transfer at low ionic strength but increased the rate at high ionic strength. This increase was larger for P. laminosum cytochrome f than for P. laminosumplastocyanin. To identify molecular features of P. laminosum cytochrome f that contribute to the increase, the effect of mutations in the N-terminal heme-shielding peptide on the reaction with P. laminosum plastocyanin was determined. Phenylalanine-3 was converted to valine and tryptophan-4 to phenylalanine or leucine. The mutations lowered the rate constant at 0.1 M ionic strength by factors of 0.71 for F4V, 0.42 for W4F, and 0.63 for W4L while introducing little change in the shape of the ionic strength dependence curve. When the N-terminal tetrapeptide (sequence YPFW) was converted into that found in the chloroplast of Chlamydomonas reinhardtii (YPVF), the reaction was slowed further (factor of 0.26). The N-terminal heme-shielding peptide was found to be responsible for 75% of the kinetic differences between cytochrome f from chloroplasts and the cyanobacterium when electrostatic interactions were eliminated.     

Roles of noncoordinated aromatic residues in redox regulation of cytochrome c3 from Desulfovibrio vulgaris Miyazaki F

The roles of aromatic residues in redox regulation of cytochrome c(3) were investigated by site-directed mutagenesis at every aromatic residue except for axial ligands (Phe20, Tyr43, Tyr65, Tyr66, His67, and Phe76). The mutations at Phe20 induced large chemical shift changes in the NMR signals for hemes 1 and 3, and large changes in the microscopic redox potentials of hemes 1 and 3. The NMR signals of the axial ligands of hemes 1 and 3 were also affected. Analysis of the nature of the mutations revealed that a hydrophobic environment and aromaticity are important for the reduction of the redox potentials of hemes 1 and 3, respectively. There was also a global effect. The replacement of Tyr66 with leucine induced chemical shift changes for heme 4, and changes in the microscopic redox potentials of heme 4. The mutations of Tyr65 induced changes in the chemical shifts and microscopic redox potentials for every heme, suggesting that Tyr65 stabilizes the global conformation, thereby reducing the redox potentials. In contrast, although the mutations of His67 and Phe76 caused chemical shift changes for heme 2, they did not affect its redox potentials, showing these residues are not important. All noncoordinated aromatic residues conserved in the cytochrome c(3) subfamily with heme binding motifs CXXCH, CXXXXCH, CXXCH, and CXXXXCH (Phe20, Tyr43, and Tyr66) are involved in the pi-pi interaction, which causes a decrease in the redox potential of the interacting heme. The global effect can be attributed to either direct or indirect interactions among the four hemes in the cyclic architecture.     

Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.     

On the structural role of the aromatic residue environment of the chlorophyll a in the cytochrome b6f complex

Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.     

Spectral components of the α-band of cytochrome oxidase

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.     

Spectral components of detergent-solubilized bovine cytochrome oxidase

Cytochrome oxidase is the terminal oxidase of the mitochondrial electron transport chain and pumps 4 protons per oxygen reduced to water. Spectral shifts in the α-band of heme a have been observed in multiple studies and these shifts have the potential to shed light on the proton pumping intermediates. Previously we found that heme a had two spectral components in the α-band during redox titrations in living RAW 264.7 mouse macrophage cells, the classical 605?nm form and a blue-shifted 602?nm form. To confirm these spectral changes were not an artifact due to the complex milieu of the living cell, redox titrations were performed in the isolated detergent-solubilized bovine enzyme from both the Soret- and α-band using precise multiwavelength spectroscopy. This data verified the presence of the 602?nm form in the α-band, revealed a similar shift of heme a in the Soret-band and ruled out the reversal of calcium binding as the origin of the blue shift. The 602?nm form was found to be stabilized at high pH or by binding of azide, which is known to blue shift the α-band of heme a. Azide also stabilized the 602?nm form in the living cells. It is concluded there is a form of cytochrome oxidase in which heme a undergoes a blue shift to a 602?nm form and that redox titrations can be successfully performed in living cells where the oxidase operates in its authentic environment and in the presence of a proton motive force.     

Plastocyanin cytochrome f interaction

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.     

Photosynthetic water oxidation in cytochrome b(559) mutants containing a disrupted heme-binding pocket

The role of cytochrome b(559) in photosynthetic oxygen evolution has been investigated in three chloroplast mutants of Chlamydomonas reinhardtii, in which one of the two histidine axial ligands to the heme, provided by the alpha subunit, has been replaced by the residues methionine, tyrosine, and glutamine. Photosystem two complexes functional for oxygen evolution could be assembled in the methionine and tyrosine mutants up to approximately 15% of wild type levels, whereas no complexes with oxygen evolution activity could be detected in the glutamine mutant. PSII supercomplexes isolated from the tyrosine and methionine mutants were as active as wild type in terms of light-saturated rates of oxygen evolution but in contrast to wild type contained no bound heme despite the presence of the alpha subunit. Oxygen evolution in the tyrosine and methionine mutants was, however, more sensitive to photoinactivation than the WT. Overall, these data establish unambiguously that a redox role for the heme of cytochrome b(559) is not required for photosynthetic oxygen evolution. Instead, our data provide new evidence of a role for cytochrome b(559) in the protection of the photosystem two complex in vivo.     

pH dependence of heme electrochemistry in cytochromes investigated by multiconformation continuum electrostatic calculations

Cytochromes belong to a diverse family of heme-containing redox proteins that function as intermediaries in electron transfer chains. They can be soluble, extrinsic, or intrinsic membrane proteins, and are found in different structural motifs (globin, 4-helix bundles, alpha beta roll, beta sandwich). Measured electrochemical midpoint potentials vary over a wide range even though the basic redox reaction at the heme is the same for all cytochromes. The perturbation of the heme electrochemistry is induced by the protein structure. Also, the pH dependence varies since it depends on the strength of interaction between the heme and surrounding residues as well as the ionization states of these groups. Multiconformation continuum electrostatics (MCCE) has been used to investigate the pH dependence of heme electrochemistry in cytochromes with different folds. Often propionates are the primary contributors for pH dependence especially if they are partially protonated in the reduced heme as it is shown for globin cytochrome c551 P. aeruginosa and cytochrome b5 R. norvegicus (alpha beta roll). However, if the propionates are already fully ionized at a certain pH they do not contribute to the pH dependence even if they have big interaction with the heme. At pH 7 there is no propionate contribution for cytochrome f C. reinhardtii (beta sandwich) and the 4-helix bundle c' R. palustris. Other residues can also change their ionization significantly during heme oxidation and therefore be involved in proton release and pH dependence. These residues have been identified for different cytochrome types.     

NMR study of the structural characteristics of variants of yeast iso-1-cytochrome c in which unvaried aromatic residues have been substituted.

The structures of variants of yeast iso-1-cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild-type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74. The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.     

Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.     

Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of Trp457.

To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS(ox); residues 1-498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) binding site located at the dimer interface and in supporting H(4)B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS(ox) dimers (residues 66-498). In W457F iNOS(ox), all the important hydrogen-bonding and aromatic stacking interactions that constitute the H(4)B binding site and that bridge the H(4)B and heme sites are preserved. In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H(4)B. Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H(4)B binding segment (Ser112-Met114), which might indicate reduced stability of the Trp457 mutant dimers. The Trp457 mutant structures show decreased pi-stacking with bound pterin when the wild-type pi-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A. The reduced pterin pi-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H(4)B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous-dioxy (Fe(II)O(2)) species during catalysis. These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H(4)B binding site and of H(4)B-bound dimers, and in influencing the rate of electron transfer between H(4)B and heme in NOS catalysis.     

Structure of the soluble domain of cytochrome f from the cyanobacterium Phormidium laminosum.

Cytochrome f from the photosynthetic cytochrome b(6)f complex is unique among c-type cytochromes in its fold and heme ligation. The 1. 9-A crystal structure of the functional, extrinsic portion of cytochrome f from the thermophilic cyanobacterium Phormidium laminosum demonstrates that an unusual buried chain of five water molecules is remarkably conserved throughout the biological range of cytochrome f from cyanobacteria to plants [Martinez et al. (1994) Structure 2, 95-105]. Structure and sequence conservation of the cytochrome f extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity of the transmembrane helix anchor. The electrostatic surface potential is variable, so that the surface of P. laminosum cytochrome f is much more acidic than that from turnip. Cytochrome f is unrelated to cytochrome c(1), its functional analogue in the mitochondrial respiratory cytochrome bc(1) complex, although other components of the b(6)f and bc(1) complexes are homologous. Identical function of the two complexes is inferred for events taking place at sites of strong sequence conservation. Conserved sites throughout the entire cytochrome b(6)f/bc(1) family include the cluster-binding domain of the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side of the membrane within the b cytochrome, but not the putative quinone-binding site on the electrochemically negative side.     

Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation.

We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., Fernandez-Velasco, J. G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b(6)f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu(29)-Gln(30)-Leu(31) sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an alpha-band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.     

Deeply branching c6-like cytochromes of cyanobacteria

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.     

Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH

Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7, 8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676), Trp(678), Phe(691), His(692), and Tyr(706), together with the neighboring Arg(414) residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH. Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/l-Arg binding and/or electron transfer to the heme from NADPH. The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(414) to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH. Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/l-Arg binding and/or efficient electron transfer to occur.     

Unfolding the unique c-type heme protein, Chlamydomonas reinhardtii cytochrome f

We have studied the unfolding reaction of cytochrome f from the green alga Chlamydomonas reinhardtii. Cytochrome f is different from all other c-type heme proteins in that it is a large, two-domain protein with predominantly beta-sheet structure. Moreover, the sixth axial ligand to the heme-iron is unique in cytochrome f: it is provided by the N-terminal alpha-amino group. Unfolding of oxidized and reduced cytochrome f by guanidine hydrochloride (GuHCl) was monitored by far-UV circular dichroism (CD), Soret absorption, and tyrosine emission: the same unfolding curves were obtained regardless of method. Neither oxidized nor reduced unfolded cytochrome f can be refolded at neutral pH. At pH 3.5 refolding takes place (upon dilution to lower denaturant concentrations or by electron injection to the unfolded, oxidized form), although the reaction is extremely slow. Reduced cytochrome f appears much more resistant towards denaturant perturbation than the oxidized form (in pH range 7-3.5). The heme in unfolded cytochrome f remains low-spin to pH 4 but turns high-spin at pH 3.5 (presumably due to protonation of the N-terminal amino group). Our results suggest that the unfolding process for cytochrome f is complex, involving kinetically trapped intermediates not resolvable by spectroscopy.     

Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.     

Analysis of the spectra and redox properties of pure cytochromes aa3.

The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.