Colin R. Andrew

Unprecedented Proximal Binding of Nitric Oxide to Heme: Implications for Guanylate Cyclase

Microbial cytochromes c′ contain a 5‐coordinate His‐ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 Å resolution crystal structures of the CO‐ and NO‐bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His–Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5‐coordinate species. In contrast, CO binds 6‐coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c′ are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.

Molecular Basis for Nitric Oxide Dynamics and Affinity with Alcaligenes xylosoxidans Cytochrome ć

The bacterial heme protein cytochrome ć from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 ± 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.

Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c–NO and 6c–CO complexes but do not form O2 complexes. Structure of the AxCYTcp–CO complex at 1.25 Å resolution shows that CO binds in two conformations moderated by the extent of displacement of the distal residue Leu16 toward the haem 7-propionate. The presence of two CO conformations is confirmed by cryogenic resonance Raman data. The preferred linear Fe–C–O arrangement (170 ± 8°) is accompanied by a flip of the propionate from the distal to proximal face of the haem. In the second conformation, the Fe–C–O unit is bent (158 ± 8°) with no flip of propionate. The energetic cost of the CO-induced Leu-propionate movements is reflected in a 600 mV (57.9 kJmol-1) decrease in haem potential, a value in good agreement with density functional theory calculations. Substitution of Leu by Ala or Gly (structures determined at 1.03 and 1.04 Å resolutions) resulted in a haem site that binds CO in the linear mode only and where no significant change in redox potential is observed. Remarkably, these variants were isolated as ferrous 6c–CO complexes, attributable to the observed eight orders of magnitude increase in affinity for CO, including an approximately 10,000-fold decrease in the rate of dissociation. These new findings have wide implications for preventing CO poisoning of gas-binding haem proteins.

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

Abstract In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on 34S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm–1 are consistent with square-planar coordination. Addition of exogenous15N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with 15N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with 15N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone.

Rebinding of Proximal Histidine in the Cytochrome c' from Alcaligenes xylosoxidans Acts as a Molecular Trap for Nitric Oxide

Transient absorption spectra on cytochrome c’ and their kinetics were recorded to identify the formation of 5-coordinate (5c)-NO and 5c-His hemes from 4c-heme (99% and 1% amplitudes; 7-ps and 100-ps time constants, respectively). We demonstrate that proximal histidine precludes NO rebinding at the proximal site.

Mechanisms of ligand discrimination in cytochromec

The gas-binding heme protein cytochrome c’ discriminates between nitric oxide (NO) and carbon monoxide (CO) while excluding the binding of molecular oxygen. In the absence of gaseous ligands, the heme Fe is 5 coordinate with a proximal histidine ligand and a vacant distal coordination site. CO binds at the distal face to form a 6-coordinate (6c) adduct, while NO forms a stable 5-coordinate (5c) proximal adduct, involving displacement of the proximal histidine, via 6c distal and transient dinitrosyl intermediates. The 6to 5coordinate conversion of NO ligation in cytochrome c’ has recently been confirmed to be highly relevant to the mechanism of activation of soluble guanylate cyclase. Using site directed mutagenesis, biophysical characterisation and correlated crystallography with single crystal resonance Raman spectroscopy we have investigated the mechanisms by which this remarkable example of ligand specificity is controlled and regulated.

Understanding the NO-Sensing Mechanism at Molecular Level

We present here how ultrafast time-resolved spectroscopy improves our understanding of a new class of proteins: Nitric Oxide sensors. Nitric oxide (NO) is a small, short-lived, and highly reactive gaseous molecule and it acts as a second messenger in several physiological systems. NO sensors are proteins which bind NO and are able to translate this binding into a signal for mammal cells as well as in bacteria. We have studied NO-sensors with the goal of understanding the activation and deactivation mechanism of the human NO-receptor, the enzyme guanylate cyclase (sGC), which is involved in communication between cells. Some bacterial sensors of NO (SONO) have structural homologies and common properties with sGC, but also have differences with sGC which make them valuable system to get structural and physiological information on sGC. To understand how NO-sensors interact with NO and control its reactivity, it is essential to probe dynamics and interactions when NO is present within protein core and what are the associated structural changes. For this purpose, we have used time-resolved absorption spectroscopy in the picoseconds (10− 12s) time domain. NO can be photodissociated from heme by the pulse of femtosecond laser. Time-resolved transient absorption spectra on NO-sensors were recorded and NO-protein interacttion were recorded. In case of cytochrome c’, we identified the formation of 5-coordinate (5c)-NO and 5c-His hemes from 4c-heme and demonstrate that proximal histidine precludes NO rebinding at the proximal site. In bacteria, the adaptation of SONO to temperature changes was not achieved by a simple temperature-dependent NO binding equilibrium, but by a change of the proportion between 5c-NO and 6c-NO species. This amplifies the response to temperature changes since a fast NO rebinding is the only property of a 5c-NO leading to 4c-heme after dissociation. Our results of NO dynamics provide a model for the regulation at molecular level in NO-sensing function.