Emma K. Wilson

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd1 nitrite reductase from P. aeruginosa.

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

How did it all start

How did life begin? Its a big question, an important aspect of which is understanding how vital biological molecules were synthesized under pre-biotic conditions. This has been an active area of research for years and a number of scenarios have been postulated. Now, Ernesto Di Mauro and colleagues (University of Rome ‘La Sapienza’) have proposed a scenario that builds on hydrogen cyanate (HCN) chemistry. They have shown that purine and pyrimidine derivatives can be synthesized directly from formamide (formed by the hydrolysis of HCN) in the presence of various metallic oxides at a fairly low temperature (160°C) [Bioorg. Med. Chem. Lett. (in press)]. This important study is the first to demonstrate that cytosine can be formed from formamide and, owing to the mild chemical conditions of the formation, has important implications for nucleotide stability. EW

Studies on Pseudomonas aeruginosa cd1 nitrite reductase: The association and dissociation reactions of the d1-heme

The dissociation and association reactions of the d1-heme, the prosthetic group characteristic of cd1 nitrite reductases (NiRs), have been investigated to assess the stability of the native enzyme. At pH 5.0 and 37 °C, the rate constant for the dissociation of the ferric d1-heme from native NiR purified from Pseudomonas aeruginosa is 4.7 ± 1.4 × 10−4 s−1. However, when the d1-heme is in the ferrous state no dissociation is observed, consistent with the shortening and strengthening of the proximal bond upon reduction of the iron. Recombinant wild-type protein and two single point mutants (Y10N and Y10F), which are expressed as semi-apo proteins and were reconstituted with synthetic d1-heme, display the same slow dissociation rate as the native enzyme. Therefore the stability of the d1-heme bound in the crevice provided by the eight-bladed s propeller domain is not altered by the act of reconstitution or by these two point mutations. The association reaction between the ferric d1-heme and semi-apo NiR is second-order and governed by an apparent rate constant of 3.3 × 106 M−1 s−1 at neutral pH and 25 °C. Interestingly, the combination rate constant is an order of magnitude slower than that reported for iron protoporphyrin IX and apomyoglobins or apohemoglobins. This difference appears to be a property of the d1-heme and not of the protein since association rate constants of CO-protoheme-Fe(II) and dicyanoprotoheme-Fe(III) with semi-apo NiR are 5 × 107 M−1 s−1 and 6 × 107 M−1 s−1, respectively. These results are discussed with reference to the structure of the d1-heme binding site, as inferred from the known 3D structure of P. aeruginosa NiR.

Welcome to the new TiBS layout

This section includes the popular features of TiBS such as Computer Corner, Book Reviews and Historical Perspective, and now also the new Q & A articles.The success of TiBS is undoubtedly a team effort, and it is the commitment and enthusiasm of our authors, referees and the Advisory Editorial Board that has ensured that TiBS has gone from strength to strength. Another extremely important member of the team is the Editor-in-Chief who, for the past eight years, has been Tim Hunt. Tim first joined the Advisory Editorial Board of TiBS in 1979, easily making him the longest-serving member to date. The relevant skill and knowledge that Tim brought with him ensured that TiBS flourished under his leadership. Tim has been an enthusiastic and tireless ambassador for the journal, and has followed the important tradition of making TiBS fun to read –TiBS will always strive to be relevant, lively, interesting and illustrated with cartoons! However, Tim has now decided to retire from the board, and is being replaced by Jan Witkowski. Jan has been a good friend to TiBS for many years and joined the Advisory Editorial Board in 1996. Jan has a wealth of knowledge and expertise in biochemistry and molecular biology, not to mention a general enthusiasm for science, so, as I mentioned earlier, the future for TiBS certainly is bright.

Membrane proteins on the move

The Keystone Symposium on Membrane protein structure/function relationships was held on 5-11 March 2001 in Tahoe City, California, USA.