Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts

Abstract

Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn; Trp-191---Phe) and distal (Trp-51----Phe; Arg-48----Leu and Lys) side of the heme. The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects. At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1. These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine. At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)

DFT Analysis of Axial and Equatorial Effects on Heme−CO Vibrational Modes: Applications to CooA and H−NOX Heme Sensor Proteins

“…The CCP-CO crystal structure shows a water molecule H-bonded to a distal Arg residue, and in turn polarizing the bound CO (28), an interaction somewhat weaker than that of H64 in Mb. Abolition of the proximal H-bond via Asp replacement in CCPMI (CCPMI(D235N)) (27) produces a positive displacement from the Mb line, consistent with moderate proximal His Hbonding (to a Ser hydroxyl group and to a backbone carbonyl) in Mb (diagram in Figure 2). A horizontal line through this point intersects the Mb line well above the WT protein, suggesting that the bound CO interacts directly with the distal Arg in CCPMI(D235N), producing a stronger distal interaction (27).…”

“…νCO was markedly lowered, while νFeC showed little change. In the case of cytochrome c peroxidase (CCPMI -a genetic variant), the effect of the strong His-Asp Hbond is evident in a large negative displacement from the Mb line (27) (Figure 2). If one draws a horizontal line through this point, it intercepts the Mb line slightly lower than the position of wild-type Mb, in which the distal histidine (H64) provides positive polarity to the bound CO.…”

Activation of Heme-regulated Eukaryotic Initiation Factor 2α Kinase by Nitric Oxide Is Induced by the Formation of a Five-coordinate NO-Heme Complex

“…In general, the Fe-His frequency is sensitive to a number of factors, including the hydrogen-bonding status of N ␦ nitrogen, strain imposed on the axial His by the protein moiety, and the geometry of bound imidazole (47)(48)(49)(50). For example, the high Fe-His frequency at 245 cm Ϫ1 for cytochrome c peroxidase (a result of the strong hydrogen bond between the axial His and Asp 235 ) shifts to 205 cm Ϫ1 following disruption of the hydrogen bond (51,52). Mb has a weak hydrogen bond on the axial His, which displays a Fe-His frequency at around 220 cm Ϫ1 .…”

“…One heme is tightly bound to the N-terminal domain, whereas the other interacts weakly with the catalytic domain (5). Under conditions of heme deficiency, HRI becomes active and phosphorylates the ␣-subunit of eIF2 at Ser 51 . It is proposed that interaction of the second heme with the catalytic domain suppresses catalysis, whereas heme deficiency leads to dissociation of heme from the catalytic domain and initiation of catalysis.…”

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met–Tyr–Trp cross-linked adduct

“…Figure 5 shows the ν(Fe-CO) versus ν(C-O) correlation that compares KatG(Y229F) to WT KatG and to peroxidases. In general, COcomplexes with ν(Fe-CO) > 520 cm −1 and ν(C-O) < 1935 cm −1 reflect increasing distal hydrogen-bonding interactions to CO and a positively charged distal heme pocket [57].…”